Deuterated compounds and uses thereof

ABSTRACT

The present invention provides for the treatment, prevention, and/or reduction of a risk of a disease, disorder, or condition in which aldehyde toxicity is implicated in the pathogenesis, including ocular disorders, skin disorders, conditions associated with injurious effects from blister agents, and autoimmune, inflammatory, neurological and cardiovascular diseases by the use of a primary amine to scavenge toxic aldehydes, such as MDA and HNE.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 17/324,318, filed May 19, 2021; which is a continuation of U.S. patent application Ser. No. 16/720,645, filed Dec. 19, 2019, now U.S. Pat. No. 11,046,650; which is a continuation of U.S. patent application Ser. No. 15/754,065, filed Feb. 21, 2018, now U.S. Pat. No. 10,550,085; which is a § 371 National Stage of PCT International Application No. PCT/US2016/048054, filed Aug. 22, 2016; which claims the benefit of U.S. Provisional Patent Application No. 62/208,223, filed Aug. 21, 2015. The entirety of each application is incorporated herein by reference thereto.

BACKGROUND OF THE INVENTION

Metabolic and inflammatory processes in cells generate toxic aldehydes, such as malondialdehyde (MDA) and 4-hydroxyl-2-nonenal (HNE or 4HNE). These aldehydes are highly reactive to proteins, carbohydrates, lipids and DNA, leading to chemically modified biological molecules, activation of inflammatory mediators such as NF-kappaB, and damage in diverse organs. For example, retinaldehyde can react with phosphatidylethanolamine (PE) to form a highly toxic compound called A2E, which is a component of lipofuscin believed to be involved in the development and progression of Age Related Macular Degeneration (AMD). Many bodily defense mechanisms function to remove or lower the levels of toxic aldehydes. Novel small molecule therapeutics can be used to scavenge “escaped” retinaldehyde in the retina, thus reducing A2E formation and lessening the risk of AMD (Jordan et al. (2006)).

Aldehydes are implicated in diverse pathological conditions such as dry eye, cataracts, keratoconus, Fuch's endothelial dystrophy in the cornea, uveitis, allergic conjunctivitis, ocular cicatricial pemphigoid, conditions associated with photorefractive keratectomy (PRK) healing or other corneal healing, conditions associated with tear lipid degradation or lacrimal gland dysfunction, inflammatory ocular conditions such as ocular rosacea (with or without meibomian gland dysfunction), and non-ocular disorders or conditions such as skin cancer, psoriasis, contact dermatitis, atopic dermatitis, acne vulgaris, Sjogren-Larsson Syndrome, ischemic-reperfusion injury, inflammation, diabetes, neurodegeneration (e.g., Parkinson's disease), scleroderma, amyotrophic lateral sclerosis, autoimmune disorders (e.g., lupus), cardiovascular disorders (e.g., atherosclerosis), and conditions associated with the injurious effects of blister agents (Negre-Salvagre et al. (2008), Nakamura et al. (2007), Batista et al. (2012), Kenney et al. (2003), Int J Dermatol 43: 494 (2004), Invest Ophthalmol Vis Sci 48: 1552 (2007), Graefe's Clin Exp Ophthalmol 233: 694 (1994), Molecular Vision 18: 194 (2012)). Reducing or eliminating aldehydes should thus ameliorate the symptoms and slow the progression of these pathological conditions.

MDA, HNE and other toxic aldehydes are generated by a myriad of metabolic mechanisms involving: fatty alcohols, sphingolipids, glycolipids, phytol, fatty acids, arachadonic acid metabolism (Rizzo (2007)), polyamine metabolism (Wood et al. (2006)), lipid peroxidation, oxidative metabolism (Buddi et al. (2002), Zhou et al. (2005)), and glucose metabolism (Pozzi et al. (2009)). Aldehydes can cross link with primary amino groups and other chemical moieties on proteins, phospholipids, carbohydrates, and DNA, leading in many cases to toxic consequences, such as mutagenesis and carcinogenesis (Marnett (2002)). MDA is associated with diseased corneas, keratoconus, bullous and other keratopathy, and Fuch's endothelial dystrophy corneas (Buddi et al. (2002)). Also, skin disorders, e.g., Sjogren-Larsson Syndrome, are likely connected with the accumulation of fatty aldehydes such as octadecanal and hexadecanal (Rizzo et al. (2010)). Further, increased lipid peroxidation and resultant aldehyde generation are associated with the toxic effects of blister agents (Sciuto et al. (2004) and Pal et al. (2009)).

There has been no suggestion in the art for treating the various conditions associated with toxic aldehydes by the administration of small molecule therapeutics acting as a scavenger for aldehydes, such as MDA and/or HNE. Thus, there is a need for treating, preventing, and/or reducing a risk of a disease or disorder in which aldehyde toxicity is implicated in the pathogenesis. The present invention addresses such a need.

Accordingly, there remains a need for treating, preventing, and/or reducing a risk of a disease, disorder, or condition in which aldehyde toxicity is implicated in the pathogenesis.

SUMMARY OF THE INVENTION

It has now been found that compounds of the present invention, and compositions thereof, are useful for treating, preventing, and/or reducing a risk of a disease, disorder, or condition in which aldehyde toxicity is implicated in the pathogenesis. Such compounds have general formula I:

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is as defined herein.

Compounds of the present invention, and pharmaceutically acceptable compositions thereof, are useful for treating a variety of diseases, disorders or conditions, associated with toxic aldehydes. Such diseases, disorders, or conditions include those described herein.

Compounds provided by this invention are also useful for the study of certain aldehydes in biology and pathological phenomena.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows overlays of EICs (extracted ion chromatograms) of the metabolite profiles of NS2 at 0 min and 120 min in human hepatocytes compared with a reference (blank hepatocytes). As the overlays show, after 120 min NS2 is metabolized to M1, M7, M8, and a low amount of M9, with some unchanged NS2 remaining.

FIG. 2 shows overlays of EICs of the metabolite profiles of NS2 at 0 min and 120 min in monkey hepatocytes compared with a reference (blank hepatocytes). As the overlays show, after 120 min NS2 is metabolized to M1, M3, M4, M7, M8, and a low amount of M9, with some unchanged NS2 remaining.

FIG. 3 shows overlays of EICs of the metabolite profiles of NS2 at 0 min and 120 min in dog hepatocytes compared with a reference (blank hepatocytes). As the overlays show, after 120 min NS2 is metabolized to M1, M2, M5, and M6, with some unchanged NS2 remaining.

FIG. 4 shows overlays of EICs of the metabolite profiles of NS2 at 0 min and 120 min in rat hepatocytes compared with a reference (blank hepatocytes). As the overlays show, after 120 min NS2 is metabolized to M1, M7, and M8, with some unchanged NS2 remaining.

FIG. 5 shows overlays of EICs of the metabolite profiles of Deuterated NS2 (NS2-D6; compound I-1) at 0 min and 120 min in human hepatocytes compared with a reference (blank hepatocytes). As the overlays show, after 120 min NS2-D6 is metabolized to a small amount of M1, with mostly unchanged NS2-D6 remaining.

FIG. 6 shows mass spectral analysis of NS2 (m/z=237).

FIG. 7 shows mass spectral analysis of metabolite M1 (m/z=253, RT=2.1 min).

FIG. 8 shows mass spectral analysis of metabolite M2 (m/z=253, RT=2.9 min).

FIG. 9 shows mass spectral analysis of metabolite M3 (m/z=429, RT=3.0 min).

FIG. 10 shows mass spectral analysis of metabolite M4 (m/z=429, RT=3.2 min).

FIG. 11 shows mass spectral analysis of metabolite M5 (m/z=542, RT=3.5 min).

FIG. 12 shows mass spectral analysis of metabolite M6 (m/z=542, RT=3.7 min). Note: m/z of 413 represents a neutral loss (NL) of 129, indicative of GSH fragmentation pattern.

FIG. 13 shows mass spectral analysis of metabolite M7 (m/z=429, RT=3.7 min).

FIG. 14 shows mass spectral analysis of metabolite M8 (m/z=413, RT=3.9 min).

FIG. 15 shows mass spectral analysis of metabolite M9 (m/z=429, RT=3.9 min).

FIG. 16 shows mass spectral analysis of NS2-D6 (compound I-1; m/z=243).

FIG. 17 shows mass spectral analysis of metabolite 1 of NS2-D6 (m/z=259).

FIG. 18 shows the effect of NS2 (CoreRx) on neuronal viability after co-treatment with 10 μM hydrogen peroxide for 5 hours.

FIG. 19 shows a graph fitting the CFDA (in relative fluorescence units) data at varying NS2 (CoreRx; in Captisol®) concentrations to a curve from which is derived the EC₅₀ value.

FIG. 20 shows the effect of NS2 (CoreRx) on cell death in hippocampal cultures after co-treatment with 10 μM hydrogen peroxide for 5 hours. *indicates data points that are significantly different from HP treatment alone.

FIG. 21 shows a graph fitting the propidium iodide data (in relative fluorescence units) at varying NS2 (CoreRx; in Captisol®) concentrations to a curve from which the EC₅₀ value is derived.

FIG. 22 shows the effect of NS2 (CoreRx; in DMSO) on neuronal viability after co-treatment with 10 mM hydrogen peroxide for 5 hours. *indicates data points that are significantly different from HP treatment alone.

FIG. 23 shows a graph fitting the CFDA (in relative fluorescence units) data at varying NS2 (CoreRx; in DMSO) concentrations to a curve from which is derived the EC₅₀ value.

FIG. 24 shows the effect of NS2 (CoreRx; in DMSO) on cell death in hippocampal cultures after co-treatment with 10 μM hydrogen peroxide. *indicates data points that are significantly different from HP treatment alone.

FIG. 25 shows a graph fitting the propidium iodide date (in relative fluorescence units) at varying NS2 (CoreRx; in DMSO) concentrations to a curve from which the EC₅₀ value is derived.

FIG. 26 shows dose response data for non-milled NS2 (J-Star) in Captisol® demonstrating the effect on neuronal viability after co-treatment with 10 μM hydrogen peroxide. *indicates data points that are significantly different from HP treatment alone.

FIG. 27 shows a graph fitting the CFDA (in relative fluorescence units) data at varying NS2 (J-Star; in DMSO) concentrations to a curve from which is derived the EC₅₀ value.

FIG. 28 shows dose response data for non-milled NS2 (J-Star) in Captisol® demonstrating the effect on cell death in hippocampal cultures after co-treatment with 10 μM hydrogen peroxide. *indicates data points that are significantly different from HP treatment alone.

FIG. 29 shows a graph fitting the propidium iodide data (in relative fluorescence units) at varying NS2 (non-milled (J-Star) in Captisol®) concentrations to a curve from which the EC₅₀ value is derived.

FIG. 30 shows dose response data for non-milled NS2 (J-Star) in DMSO demonstrating the effect on neuronal viability in hippocampal cultures after co-treatment with 10 μM hydrogen peroxide. *indicates data points that are significantly different from HP treatment alone.

FIG. 31 shows a graph fitting the CFDA (in relative fluorescence units) data at varying non-milled NS2 ((J-Star) in DMSO) concentrations to a curve from which is derived the EC₅₀ value.

FIG. 32 shows dose response data for non-milled NS2 (J-Star) in DMSO showing effect on cell death in hippocampal cells after co-treatment with 10 μM hydrogen peroxide. *indicates data points that are significantly different from HP treatment alone.

FIG. 33 shows a graph fitting the propidium iodide data (in relative fluorescence units) at varying non-milled NS2 ((J-Star) in DMSO) concentrations to a curve from which the EC₅₀ value is derived.

FIG. 34 shows dose response data for the formulation vehicles on neuronal cell viability after co-treatment with 10 μM hydrogen peroxide.

FIG. 35 shows dose response data for the formulation vehicles on cell death after co-treatment with 10 μM hydrogen peroxide.

FIG. 36 shows the effect of ALD-6 (compound I-1) on neuronal viability in hippocampal cultures treated with 10 μM hydrogen peroxide. * indicates data points that are significantly different from HP treatment alone.

FIG. 37 shows the calculated logistic curve and EC₅₀ value for neuronal viability assay using hippocampal cultures treated with ALD-6.

FIG. 38 shows dose response effect of ALD-6 on cell death after co-treatment with 10 μM hydrogen peroxide. *indicates data points that are significantly different from HP treatment alone.

FIG. 39 shows the calculated logistic curve and EC₅₀ value for cell death assay using hippocampal cultures treated with ALD-6.

FIG. 40 shows dose response effect of ALD-5 on neuronal viability after co-treatment with 10 μM hydrogen peroxide.

FIG. 41 shows dose response effect of ALD-5 on cell death after co-treatment with 10 μM hydrogen peroxide.

FIG. 42 shows dose response effect of ALD-2 on neuronal viability after co-treatment with 10 μM hydrogen peroxide.

FIG. 43 shows dose response effect of ALD-2 on cell death after co-treatment with 10 μM hydrogen peroxide.

FIGS. 44-46 show a histogram of specific binding results for NS2-D6 expressed as a percentage of the specific binding of each control compound.

FIG. 47 shows a histogram of in vitro pharmacology results in enzyme and uptake assays for NS2-D6.

DETAILED DESCRIPTION OF THE INVENTION 1. General Description of Certain Aspects of the Invention

In certain embodiments, the present invention provides compounds, compositions, and methods for treatment, prevention, and/or reduction of a risk of diseases, disorders, or conditions in which aldehyde toxicity is implicated in the pathogenesis. In some embodiments, such compounds include those of the formulae described herein, or a pharmaceutically acceptable salt thereof, wherein each variable is as defined herein and described in embodiments. In one aspect, the present invention provides a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R¹ is selected from —NH₂, —NHD, or —ND₂;     -   R² is selected from hydrogen or deuterium;     -   R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or         —CD₃; and     -   R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen         or deuterium;     -   provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸         is or contains deuterium.

2. Definitions

Compounds of this invention include those described generally above, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N⁺(C₁₋₄ alkyl)₄ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.

Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

The “retina” is a region of the central nervous system with approximately 150 million neurons. It is located at the back of the eye where it rests upon a specialized epithelial tissue called retinal pigment epithelium (RPE). The retina initiates the first stage of visual processing by transducing visual stimuli in specialized neurons called “photoreceptors”. Their synaptic outputs are processed by elaborate neural networks in the retina and are then transmitted to the brain. The retina has evolved two specialized classes of photoreceptors to operate under a wide range of light conditions. “Rod” photoreceptors transduce visual images under low light conditions and mediate achromatic vision. “Cone” photoreceptors transduce visual images in dim to bright light conditions and mediate both color vision and high acuity vision.

Every photoreceptor is compartmentalized into two regions called the “outer” and “inner” segment. The inner segment is the neuronal cell body containing the cell nucleus. The inner segment survives for a lifetime in the absence of retinal disease. The outer segment is the region where the light sensitive visual pigment molecules are concentrated in a dense array of stacked membrane structures. Part of the outer segment is routinely shed and regrown in a diurnal process called outer segment renewal. Shed outer segments are ingested and metabolized by RPE cells.

The “macula” is the central region of the retina which contains the fovea where visual images are processed by long slender cones in high spatial detail (“visual acuity”). “Macular degeneration” is a form of retinal neurodegeneration which attacks the macula and destroys high acuity vision in the center of the visual field. Age-Related Macular Degeneration (AMD) begins in a “dry form” characterized by residual lysosomal granules called lipofuscin in RPE cells, and by extracellular deposits called “drusen”. Drusen contain cellular waste products excreted by RPE cells. “Lipofuscin” and drusen can be detected clinically by ophthalmologists and quantified using fluorescence techniques. They can be the first clinical signs of macular degeneration.

Lipofuscin contains aggregations of A2E. Lipofuscin accumulates in RPE cells and poisons them by multiple known mechanisms. As RPE cells become poisoned, their biochemical activities decline and photoreceptors begin to degenerate. Extracellular drusen may further compromise RPE cells by interfering with their supply of vascular nutrients. Drusen also trigger inflammatory processes, which lead to choroidal neovascular invasions of the macula in one patient in ten who progresses to wet form AMD. Both the dry form and wet form progress to blindness.

“ERG” is an acronym for electroretinogram, which is the measurement of the electric field potential emitted by retinal neurons during their response to an experimentally defined light stimulus. ERG is a non-invasive measurement which can be performed on either living subjects (human or animal) or a hemisected eye in solution that has been removed surgically from a living animal.

As used herein, the term “RAL” means retinaldehyde. The term “RAL-trap” means a therapeutic compound that binds free RAL and thereby prevents the RAL from Schiff base condensation with membrane phosphatidylethanolamine (PE). “Free RAL” is defined as RAL that is not bound to a visual cycle protein. The terms “trans-RAL” and “all-trans-RAL” are used interchangeably and mean all trans-retinaldehyde.

A2E is a reaction by-product of a complex biochemical pathway called the “visual cycle” which operates collaboratively in both RPE cells and photoreceptor outer segments. The visual cycle recycles a photoreactive aldehyde chromophore called “retinaldehyde” which is derived from vitamin A and is essential for vision. In simplified terms, the visual cycle has four principal steps: 1) it converts vitamin A in the RPE into an aldehyde chromophore with one photoreactive strained double bond (11-cis-RAL); 2) it transports 11-cis-RAL to the retina where it binds to a specialized photoreceptor protein called opsin; 3) light photoisomerizes bound 11-cis-RAL to trans-RAL, which initiates the release of bound RAL from the opsin binding site; and 4) it converts trans-RAL (an aldehyde) to vitamin A (an alcohol) and transports vitamin A back to the RPE where the cycle begins again.

The aldehyde group of RAL helps bind the molecule to opsin by forming a reversible chemical bond to an amino acid sidechain in the opsin binding site. While the aldehyde group on RAL is essential for anchoring the molecule to the opsin binding site, it is otherwise hazardous because of its propensity to form Schiff bases with other biological amines. The first three reactions take place in photoreceptor outer segments and produce an intermediary product called A2PE. Once formed, A2PE partitions into the lipid phase and accumulates in photoreceptor outer segment membranes. When RPE cells ingest discarded outer segments, their accumulated A2PE is routed to their lysosomes.

As described above, macular degeneration and other forms of retinal disease whose etiology involves the accumulation of A2E and/or lipofuscin may be treated or prevented by lowering the amount of A2E formed. Compounds useful for doing so include RAL-traps. RAL-traps lower the amount of A2E formed, for example by forming a covalent bond with RAL that has escaped sequestering. RAL that has reacted with a RAL-trap compound is thereby unavailable to react with phosphatidyl ethanolamine.

The present invention is also directed to the use of a compound described herein in the manufacture of a medicament for the treatment, prevention, and/or reduction of a risk of a disease, disorder, or condition in which aldehyde toxicity is implicated in the pathogenesis. More specifically this aspect of the invention is directed to the use of a compound described herein in the manufacture of a medicament for the treatment, prevention, and/or reduction of a risk of (1) an ocular disease, disorder, or condition, including, but not limited to, a corneal disease (e.g., dry eye syndrome, cataracts, keratoconus, bullous and other keratopathy, and Fuch's endothelial dystrophy), other ocular disorders or conditions (e.g., allergic conjunctivitis, ocular cicatricial pemphigoid, conditions associated with PRK healing and other corneal healing, and conditions associated with tear lipid degradation or lacrimal gland dysfunction), and other ocular conditions associated with high aldehyde levels as a result of inflammation (e.g., uveitis, scleritis, ocular Stevens Johnson Syndrome, and ocular rosacea (with or without meibomian gland dysfunction)), (2) a skin disorder or condition or a cosmetic indication. For example, the disease, disorder, or condition includes, but is not limited to, psoriasis, topical (discoid) lupus, contact dermatitis, atopic dermatitis, allergic dermatitis, radiation dermatitis, acne vulgaris, Sjogren-Larsson Syndrome and other ichthyosis, solar elastosis/wrinkles, skin tone firmness, puffiness, eczema, smoke or irritant induced skin changes, dermal incision, and a skin condition associated burn and wound, (3) a condition associated with the toxic effects of blister agents or burns from alkali agents, or (4) an autoimmune, immune-mediated, inflammatory, cardiovascular, or neurological disease (e.g., lupus, scleroderma, asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, inflammatory bowel disease, sepsis, atherosclerosis, ischemic-reperfusion injury, Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, diabetes, metabolic syndrome, and fibrotic diseases).

The present invention is also directed to the use of a compound described herein in treating, preventing, and/or reducing a risk of a disease, disorder, or condition in which aldehyde toxicity is implicated in the pathogenesis. More specifically this aspect of the invention is directed to the use of a compound described herein in treating, preventing, and/or reducing a risk of (1) an ocular disease, disorder, or condition, including, but not limited to, a corneal disease (e.g., dry eye syndrome, cataracts, keratoconus, bullous and other keratopathy, and Fuch's endothelial dystrophy), other ocular disorders or conditions (e.g., allergic conjunctivitis, ocular cicatricial pemphigoid, conditions associated with PRK healing and other corneal healing, and conditions associated with tear lipid degradation or lacrimal gland dysfunction), and other ocular conditions associated with high aldehyde levels as a result of inflammation (e.g., uveitis, scleritis, ocular Stevens Johnson Syndrome, and ocular rosacea (with or without meibomian gland dysfunction)), (2) a skin disorder or condition or a cosmetic indication. For example, the disease, disorder, or condition includes, but is not limited to, psoriasis, topical (discoid) lupus, contact dermatitis, atopic dermatitis, allergic dermatitis, radiation dermatitis, acne vulgaris, Sjogren-Larsson Syndrome and other ichthyosis, solar elastosis/wrinkles, skin tone firmness, puffiness, eczema, smoke or irritant induced skin changes, dermal incision, and a skin condition associated burn and wound, (3) a condition associated with the toxic effects of blister agents or burns from alkali agents, or (4) an autoimmune, immune-mediated, inflammatory, cardiovascular, or neurological disease (e.g., lupus, scleroderma, asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, inflammatory bowel disease, sepsis, atherosclerosis, ischemic-reperfusion injury, Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, diabetes, metabolic syndrome, and fibrotic diseases).

The compounds described herein can also be administered topically, such as directly to the eye, e.g., as an eye-drop or ophthalmic ointment. Eye drops typically comprise an effective amount of at least one compound described herein and a carrier capable of being safely applied to an eye. For example, the eye drops are in the form of an isotonic solution, and the pH of the solution is adjusted so that there is no irritation of the eye. In many instances, the epithelial barrier interferes with penetration of molecules into the eye. Thus, most currently used ophthalmic drugs are supplemented with some form of penetration enhancer. These penetration enhancers work by loosening the tight junctions of the most superior epithelial cells (Burstein, Trans Ophthalmol Soc UK 104: 402 (1985); Ashton et al., J Pharmacol Exp Ther 259: 719 (1991); Green et al., Am J Ophthalmol 72: 897 (1971)). The most commonly used penetration enhancer is benzalkonium chloride (Tang et al., J Pharm Sci 83: 85 (1994); Burstein et al, Invest Ophthalmol Vis Sci 19: 308 (1980)), which also works as preservative against microbial contamination.

Topical administration may be in the form of a cream, suspension, emulsion, ointment, drops, oil, lotion, patch, tape, inhalant, spray, or controlled release topical formulations including gels, films, patches, and adhesives. Intra-ocular administration may take the form of subconjunctival, subtenon's capsule, retrobulbar or intravitreal injections, depots or implants. Compounds administered by these routes may be in solution or suspension form. Administration of compounds by depot injection may contain pharmaceutically acceptable carriers or excipients; these may be natural or synthetic and may be biodegradable or non-biodegradable and facilitate drug release in a controlled manner. Implants used for controlled release of compound may be composed of natural or synthetic, biodegradable or non-biodegradable materials. The carrier is acceptable in that it is compatible with the other components of the composition and is not injurious to the patient. Some examples of carriers include (1) sugars such as lactose glucose and sucrose, (2) starches such as corn starch and potato starch, (3) cellulose and (4) cyclodextrins. A useful topical formulation is described in PCT publication WO 2011/072141, the contents of which are herein incorporated by reference.

Formulations for topical administration to the skin can include, for example, ointments, creams, gels and pastes comprising the primary amine compound in a pharmaceutical acceptable carrier. The formulation of the primary amine compound for topical use includes the preparation of oleaginous or water-soluble ointment bases, as is well known to those in the art. For example, these formulations may include vegetable oils, animal fats, and, for example, semisolid hydrocarbons obtained from petroleum. Particular components used may include white ointment, yellow ointment, cetyl esters wax, oleic acid, olive oil, paraffin, petrolatum, white petrolatum, spermaceti, starch glycerite, white wax, yellow wax, lanolin, anhydrous lanolin and glyceryl monostearate. Various water-soluble ointment bases may also be used, including glycol ethers and derivatives, polyethylene glycols, polyoxyl 40 stearate and polysorbates.

The formulations for topical administration may contain the compound used in the present application at a concentration in the range of 0.001-10%, 0.05-10%, 0.1-10%, 0.2-10%, 0.5-10%, 1-10%, 2-10%, 3-10%, 4-10%, 5-10%, or 7-10% (weight/volume), or in the range of 0.001-2.0%, 0.001-1.5%, or 0.001-1.0%, (weight/volume), or in the range of 0.05-2.0%, 0.05-1.5%, or 0.05-1.0%, (weight/volume), or in the range of 0.1-5.0%, 0.1-2.0%, 0.1-1.5%, or 0.1-1.0% (weight/volume), or in the range of 0.5-5.0%, 0.5-2.0%, 0.5-1.5%, or 0.5-1.0% (weight/volume), or in the range of 1-5.0%, 1-2.0%, or 1-1.5% (weight/volume). The formulations for topical administration may also contain the compound used in the present application at a concentration in the range of 0.001-2.5%, 0.01-2.5%, 0.05-2.0%, 0.1-2.0%, 0.2-2.0%, 0.5-2.0%, or 1-2.0% (weight/weight), or in the range of 0.001-2.0%, 0.001-1.5%, 0.001-1.0%, or 0.001-5% (weight/weight).

In an eye drop formulation the composition may contain the active compound at a concentration of 0.01-20%, 0.02-15%, 0.04-10%, 0.06-5%, 0.08-1%, or 0.09-0.5% (weight/volume) with or without pH and/or osmotic adjustment to the solution. More particularly, the eye drop formulation may contain a compound described herein at a concentration of 0.09-0.5% (weight/volume), such as 0.1%.

In one exemplification, the pharmaceutical compositions encompass a composition made by admixing a therapeutically effective amount of a compound described herein with an oligomeric or a polymeric carrier such as a cyclodextrin, or chemically modified cyclodextrin, including trimethyl-β-cyclodextrin, 2-hydroxyethyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, 3-hydroxypropyl-β-cyclodextrin, and β-cyclodextrin sulfobutylether sodium salt (or potassium salt). Exemplifying an oligomeric or a polymeric carrier is β-cyclodextrin sulfobutylether sodium salt. The amount of β-cyclodextrin sulfobutylether sodium salt in the composition may range from about 0.01% to 30% weight/volume. In one illustration, the concentration of β-cyclodextrin sulfobutylether sodium salt is 5-25% weight/volume. Further illustrating the concentration of β-cyclodextrin sulfobutylether sodium salt is 6-20% weight/volume. In one exemplification the concentration of β-cyclodextrin sulfobutylether is 6-12% weight/volume. Further exemplifying the concentration of β-cyclodextrin sulfobutylether is 9-10% weight/volume, including 9.5% weight/volume. The amount of the compound described herein in the composition may range 0.01-20%, 0.02-15%, 0.04-10%, 0.06-5%, 0.08-1%, or 0.09-0.5% (weight/volume). More particularly, the composition may contain a compound described herein at a concentration of 0.09-0.5% (weight/volume), such as 0.1%.

The compounds described herein may be administered orally and as such the pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.

For oral administration in the form of a tablet or capsule (e.g., a gelatin capsule), the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum starches, agar, alginic acid or its sodium salt, or effervescent mixtures, croscarmellose or its sodium salt, and the like. Diluents, include, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine.

Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

A therapeutically effective dose, of a compound described herein in an oral formulation, may vary from 0.01 mg/kg to 50 mg/kg patient body weight per day, more particularly 0.01 to 10 mg/kg, which can be administered in single or multiple doses per day. For oral administration the drug can be delivered in the form of tablets or capsules containing 1 mg to 500 mg of the active ingredient specifically, 1 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, 250 mg, and 500 mg, or in the forms of tables or capsules containing at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% (w/w) of the active ingredient. For example, the capsules may contain 50 mg of the active ingredient, or 5-10% (w/w) of the active ingredient. For example, the tablets may contain 100 mg of the active ingredient, or 20-50% (w/w) of the active ingredient. For example, the tablet may contain, in addition to the active ingredient, a disintegrant (e.g., croscarmellose or its sodium salt and methyl cellulose), a diluent (e.g., microcrystalline cellulose), and a lubricant (e.g., sodium stearate and magnesium stearate). The drug can be administered on a daily basis either once, twice or more per day.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

Parenteral formulations comprising a compound described herein can be prepared in aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The formulations may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. The compositions are prepared according to conventional methods, and may contain about 0.1 to 75%, preferably about 1 to 50%, of a compound described herein.

The phrases “parenteral administration” and “administered parenterally” are art-recognized terms, and include modes of administration other than enteral and topical administration, such as injections, and include, without limitation, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

3. Description of Exemplary Compounds

Deuterium (D or ²H) is a stable, non-radioactive isotope of hydrogen and has an atomic weight of 2.0144. Hydrogen naturally occurs as a mixture of the isotopes ¹H (hydrogen or protium), D (²H or deuterium), and T (³H or tritium). One skilled in the art appreciates that the designation “hydrogen” in hydrogen-containing chemical compounds actually represents a mixture of hydrogen and about 0.015% deuterium.

Complete deuteration, or 100% deuteration, at any one site can be difficult to achieve in the laboratory. When a deuterium atom is indicated at a given site on any compound described herein, it is understood that a small percentage of hydrogen may still be present. Such compounds are said to be enriched with deuterium. Deuterium-enriched compounds are prepared via synthesis utilizing appropriately enriched starting materials. As used herein, the terms “deuterium-enriched” or “deuterium enrichment” refer to a compound, or a particular site of said compound, which comprises deuterium in an amount that is greater than its natural isotopic abundance (0.015%). Accordingly, in some embodiments, the present invention provides compounds comprising deuterium at a given site, wherein the percentage or level of deuterium incorporation is greater than its natural isotopic abundance.

According to one aspect, the present invention provides a compound of formula I:

-   -   or a pharmaceutically acceptable salt thereof, wherein:     -   R¹ is selected from —NH₂, —NHD, or —ND₂;     -   R² is selected from hydrogen or deuterium;     -   R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or         —CD₃; and     -   R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen         or deuterium;     -   provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸         is or contains deuterium.

According to another aspect, the present invention provides a compound of formula I-A:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R¹ is selected from —NH₂, —NHD, or —ND₂;     -   R² is selected from hydrogen or deuterium;     -   R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or         —CD₃; and     -   R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen         or deuterium;     -   provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸         is or contains deuterium.

According to another aspect, the present invention provides a compound of formulae II-A or II-B:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R¹ is selected from —NH₂, —NHD, or —ND₂;     -   R² is selected from hydrogen or deuterium;     -   R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or         —CD₃; and     -   R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen         or deuterium;         provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸         in formula II-A is or contains deuterium.

According to another aspect, the present invention provides a compound of formulae III-A, III-B, or III-C:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R¹ is selected from —NH₂, —NHD, or —ND₂;     -   R² is selected from hydrogen or deuterium;     -   R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or         —CD₃; and     -   R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen         or deuterium;         provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸         is or contains deuterium.

According to another aspect, the present invention provides a compound of formula IV:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R¹ is selected from —NH₂, —NHD, or —ND₂;     -   R² is selected from hydrogen or deuterium;     -   R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or         —CD₃; and     -   R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen         or deuterium;         provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸         is or contains deuterium.

According to another aspect, the present invention provides a compound of formula V:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R¹ is selected from —NH₂, —NHD, or —ND₂;     -   R² is selected from hydrogen or deuterium;     -   R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or         —CD₃; and     -   R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen         or deuterium;         provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸         is or contains deuterium.

According to another aspect, the present invention provides a compound of formulae VI-A or VI-B:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   each A is independently hydrogen or deuterium;     -   R¹ is selected from —NH₂, —NHD, or —ND₂;     -   R² is selected from hydrogen or deuterium; and     -   R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen         or deuterium;         provided that at least one of A, R¹, R², R⁵, R⁶, R⁷, or R⁸ is or         contains deuterium.

According to another aspect, the present invention provides a compound of formulae VII-A or VII-B:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   each A is independently hydrogen or deuterium;     -   R¹ is selected from —NH₂, —NHD, or —ND₂;     -   R² is selected from hydrogen or deuterium;     -   R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or         —CD₃; and     -   R⁸ is selected from hydrogen or deuterium;     -   provided that at least one of R¹, R², R³, R⁴, A, or R⁸ is or         contains deuterium.

According to another aspect, the present invention provides a compound of formulae VIII-A or VIII-B:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   each A is independently hydrogen or deuterium;     -   R¹ is selected from —NH₂, —NHD, or —ND₂;     -   R² is selected from hydrogen or deuterium;     -   R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or         —CD₃; and     -   R⁵ and R⁸ are each independently selected from hydrogen or         deuterium;         provided that at least one of A, R¹, R², R³, R⁴, R⁵, or R⁸ is or         contains deuterium.

According to another aspect, the present invention provides a compound of formula IX-A or IX-B:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R¹ is selected from —NH₂, —NHD, or —ND₂;     -   R² is selected from hydrogen or deuterium;     -   R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or         —CD₃; and     -   R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen         or deuterium;         provided that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸         is or contains deuterium.

According to another aspect, the present invention provides a compound of formula X:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R¹ is selected from —NH₂, —NHD, or —ND₂;     -   R² is selected from hydrogen or deuterium;     -   R³ and R⁴ are independently selected from —CH₃, —CH₂D, —CHD₂, or         —CD₃; and     -   R⁵ and R⁶ are each independently selected from hydrogen or         deuterium;         provided that at least one of R¹, R², R³, R⁴, R⁵, or R⁶ is or         contains deuterium.

The following embodiments are applicable to each of the preceding formulae.

As defined above and described herein, R¹ is selected from —NH₂, —NHD, or —ND₂.

In some embodiments, R¹ is —NH₂. In some embodiments, R¹ is —NH₂ and at least one of R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

In some embodiments, R¹ is —NHD. In some embodiments, R¹ is —NHD and at least one of R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

In some embodiments, R¹ is —ND₂. In some embodiments, R¹ is —ND₂ and at least one of R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

As defined above and described herein, A is selected from hydrogen or deuterium.

In some embodiments, A is hydrogen. In some embodiments, A is hydrogen and at least one of R¹, R³, R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium. In some embodiments, A is deuterium. In some embodiments, A is deuterium and at least one of R¹, R³, R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

As defined above and described herein, R² is selected from hydrogen or deuterium.

In some embodiments, R² is hydrogen. In some embodiments, R² is hydrogen and at least one of R¹, R³, R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium. In some embodiments, R² is deuterium. In some embodiments, R² is deuterium and at least one of R¹, R³, R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

As defined above and described herein, R³ is selected from —CH₃, —CH₂D, —CHD₂, or —CD₃.

In some embodiments, R³ is —CH₃. In some embodiments, R³ is —CH₃ and at least one of R¹, R², R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

In some embodiments, R³ is —CH₂D. In some embodiments, R³ is —CH₂D and at least one of R¹, R², R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

In some embodiments, R³ is —CHD₂. In some embodiments, R³ is —CHD₂ and at least one of R¹, R², R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

In some embodiments, R³ is —CD₃. In some embodiments, R³ is —CD₃ and at least one of R¹, R², R⁴, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

As defined above and described herein, R⁴ is selected from —CH₃, —CH₂D, —CHD₂, or —CD₃.

In some embodiments, R⁴ is —CH₃. In some embodiments, R⁴ is —CH₃ and at least one of R¹, R², R³, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

In some embodiments, R⁴ is —CH₂D. In some embodiments, R⁴ is —CH₂D and at least one of R¹, R², R³, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

In some embodiments, R⁴ is —CHD₂. In some embodiments, R⁴ is —CHD₂ and at least one of R¹, R², R³, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

In some embodiments, R⁴ is —CD₃. In some embodiments, R⁴ is —CD₃ and at least one of R¹, R², R³, R⁵, R⁶, R⁷, or R⁸ is or contains deuterium.

As defined above and described herein, R⁵ is selected from hydrogen or deuterium.

In some embodiments, R⁵ is hydrogen. In some embodiments, R⁵ is hydrogen and at least one of R¹, R², R³, R⁴, R⁶, R⁷, or R⁸ is or contains deuterium. In some embodiments, R⁵ is deuterium. In some embodiments, R⁵ is deuterium and at least one of R¹, R², R³, R⁴, R⁶, R⁷, or R⁸ is or contains deuterium.

As defined above and described herein, R⁶ is selected from hydrogen or deuterium.

In some embodiments, R⁶ is hydrogen. In some embodiments, R⁶ is hydrogen and at least one of R¹, R², R³, R⁴, R⁵, R⁷, or R⁸ is or contains deuterium. In some embodiments, R⁶ is deuterium. In some embodiments, R⁶ is deuterium and at least one of R¹, R², R³, R⁴, R⁵, R⁷, or R⁸ is or contains deuterium.

As defined above and described herein, R⁷ is selected from hydrogen or deuterium.

In some embodiments, R⁷ is hydrogen. In some embodiments, R⁷ is hydrogen and at least one of R¹, R², R³, R⁴, R⁵, or R⁸ is or contains deuterium. In some embodiments, R⁷ is deuterium. In some embodiments, R⁷ is deuterium and at least one of R¹, R², R³, R⁴, R⁵, R⁶, or R⁷ is or contains deuterium.

As defined above and described herein, R⁸ is selected from hydrogen or deuterium.

In some embodiments, R⁸ is hydrogen. In some embodiments, R⁸ is hydrogen and at least one of R¹, R², R³, R⁴, R⁵, R⁶, or R⁷ is or contains deuterium. In some embodiments, R⁸ is deuterium. In some embodiments, R⁸ is deuterium and at least one of R¹, R², R³, R⁴, R⁵, R⁶, or R⁷ is or contains deuterium.

In some embodiments, the present invention provides a compound of formulae I, I-A, II-A, II-B, III-A, III-B, III-C, IV, or V, wherein each of R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is as defined above and described herein, and wherein each of R¹ and R² is as defined in an entry set forth in Table 1a, below.

TABLE 1a Entry R¹ R² i —NH₂ H ii —NH₂ D iii —NHD H iv —NHD D v —ND₂ H vi —ND₂ D

In some embodiments, the present invention provides a compound of formulae I, I-A, II-A, II-B, III-A, III-B, III-C, IV, or V, wherein each of R¹, R², R⁵, R⁶, R⁷, and R⁸ is as defined above and described herein, and wherein each of R³ and R⁴ is as defined in an entry set forth in Table 1b, below.

TABLE 1b Entry R³ R⁴ i —CH₃ —CH₃ ii —CH₃ —CH₂D iii —CH₃ —CHD₂ iv —CH₃ —CD₃ v —CH₂D —CH₃ vi —CH₂D —CH₂D vii —CH₂D —CHD₂ viii —CH₂D —CD₃ ix —CHD₂ —CH₃ x —CHD₂ —CH₂D xi —CHD₂ —CHD₂ xii —CHD₂ —CD₃ xiii —CD₃ —CH₃ xiv —CD₃ —CH₂D xv —CD₃ —CHD₂ xvi —CD₃ —CD₃

In some embodiments, the present invention provides a compound of formulae I, I-A, II-A, II-B, III-A, III-B, III-C, IV, or V, wherein each of R¹, R², R³, and R⁴ is as defined above and described herein, and wherein each of R⁵, R⁶, R⁷, and R⁸ is as defined in an entry set forth in Table 1c, below.

TABLE 1c Entry R⁵ R⁶ R⁷ R⁸ i H H H H ii H H H D iii H H D H iv H D H H v D H H H vi H H D D vii H D H D viii D H H D ix H D D H x D H D H xi D D H H xii H D D D xiii D H D D xiv D D H D xv D D D H xvi D D D D

In some embodiments, the present invention provides a compound of formulae I, I-A, II-A, II-B, III-A, III-B, III-C, IV, or V, wherein each of R¹ and R² is as defined in an entry set forth in Table 1a, above, each of R³ and R⁴ is as defined in an entry set forth in Table 1b, above, and each of R⁵, R⁶, R⁷, and R⁸, is as defined in an entry set forth in Table 1c, above.

In some embodiments, the present invention provides a compound selected from those recited in any of Table 1a, Table 1b, or Table 1c, or a pharmaceutically acceptable salt thereof.

In some embodiments, present invention provides a compound of formula I selected from these depicted in Table 2, below.

TABLE 2 Representative Compounds of Formula I

I-1

I-2

I-3

I-4

I-5

I-6

I-7

I-8

I-9

I-10

I-11

I-12

I-13

I-14

I-15

I-16

I-17

I-18

I-19

I-20

I-21

I-22

I-23

I-24

I-25

I-26

I-27

I-28

I-29

I-30

I-31

In some embodiments, the present invention provides a compound depicted in Table 2, above, or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a deuterium-enriched analogue of a compound depicted in Table 2A, below, or a pharmaceutically acceptable salt thereof, in which deuterium is enriched at any available hydrogen.

TABLE 2A

I-32

I-33

I-34

I-35

I-36

I-37

I-38

I-39

I-40

I-41

I-42

I-43

I-44

I-45

I-46

I-47

In some embodiments, the present invention provides any compound described herein comprising one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen deuterium atoms.

In some embodiments, provided compounds comprise deuterium in an amount of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. As used herein in the context of deuterium enrichment, the term “about” means ±2%.

In certain embodiments, the present invention provides any compound described above and herein, or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides any compound described above and herein in isolated form.

4. Uses of Compounds and Pharmaceutically Acceptable Compositions Thereof

Certain compounds described herein are found to be useful in scavenging toxic aldehydes, such as MDA and HNE. The compounds described herein undergo a Schiff base condensation with MDA, HNE, or other toxic aldehydes, and form a complex with the aldehydes in an energetically favorable reaction, thus reducing or eliminating aldehydes available for reaction with a protein, lipid, carbohydrate, or DNA. Importantly, compounds described herein can react with aldehydes to form a compound having a closed-ring structure that contains the aldehydes, thus trapping the aldehydes and preventing the aldehydes from being released back into the cellular milieu.

As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment is administered after one or more symptoms have developed. In other embodiments, treatment is administered in the absence of symptoms. For example, treatment is administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment is also continued after symptoms have resolved, for example to prevent, delay or lessen the severity of their recurrence.

The invention relates to compounds described herein for the treatment, prevention, and/or reduction of a risk of diseases, disorders, or conditions in which aldehyde toxicity is implicated in the pathogenesis.

Examples of the diseases, disorders, or conditions in which aldehyde toxicity is implicated include an ocular disease, disorder, or condition, including, but not limited to, a corneal disease (e.g., dry eye syndrome, cataracts, keratoconus, bullous and other keratopathy, and Fuch's endothelial dystrophy), other ocular disorders or conditions (e.g., allergic conjunctivitis, ocular cicatricial pemphigoid, conditions associated with PRK healing and other corneal healing, and conditions associated with tear lipid degradation or lacrimal gland dysfunction), and other ocular conditions associated with high aldehyde levels as a result of inflammation (e.g., uveitis, scleritis, ocular Stevens Johnson Syndrome, ocular rosacea (with or without meibomian gland dysfunction)). In one example, the ocular disease, disorder, or condition is not macular degeneration, such as age-related macular degeneration (“AMD”), or Stargardt's disease. In a further example, the ocular disease, disorder, or condition is dry eye syndrome, ocular rosacea, or uveitis.

Examples of the diseases, disorders, conditions, or indications in which aldehyde toxicity is implicated also include non-ocular disorders, including psoriasis, topical (discoid) lupus, contact dermatitis, atopic dermatitis, allergic dermatitis, radiation dermatitis, acne vulgaris, Sjogren-Larsson Syndrome and other ichthyosis, solar elastosis/wrinkles, skin tone firmness, puffiness, eczema, smoke or irritant induced skin changes, dermal incision, a skin condition associated burn and/or wound, lupus, scleroderma, asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, inflammatory bowel disease, sepsis, atherosclerosis, ischemic-reperfusion injury, Parkinson's disease, Alzheimer's disease, succinic semialdehyde dehydrogenase deficiency, multiple sclerosis, amyotrophic lateral sclerosis, diabetes, metabolic syndrome, age-related disorders, and fibrotic diseases. In a further example, the non-ocular disorder is a skin disease, disorder, or condition selected from contact dermatitis, atopic dermatitis, allergic dermatitis, and. radiation dermatitis. In another example, the non-ocular disorder is a skin disease, disorder, or condition selected from Sjogren-Larsson Syndrome and a cosmetic indication associated burn and/or wound.

In a further example, the diseases, disorders, or conditions in which aldehyde toxicity is implicated are an age-related disorder. Examples of age-related diseases, disorders, or conditions include wrinkles, dryness, and pigmentation of the skin.

Examples of the diseases, disorders, or conditions in which aldehyde toxicity is implicated further include conditions associated with the toxic effects of blister agents or burns from alkali agents. The compounds described herein reduce or eliminate toxic aldehydes and thus treat, prevent, and/or reduce a risk of these diseases or disorders.

In one embodiment, the invention relates to the treatment, prevention, and/or reduction of a risk of an ocular disease, disorder, or condition in which aldehyde toxicity is implicated in the pathogenesis, comprising administering to a subject in need thereof a compound described herein. The ocular disease, disorder, or condition includes, but is not limited to, a corneal disease (e.g., dry eye syndrome, cataracts, keratoconus, bullous and other keratopathy, and Fuch's endothelial dystrophy in the cornea), other ocular disorders or conditions (e.g., allergic conjunctivitis, ocular cicatricial pemphigoid, conditions associated with PRK healing and other corneal healing, and conditions associated with tear lipid degradation or lacrimal gland dysfunction), and other ocular conditions where inflammation leads to high aldehyde levels (e.g., uveitis, scleritis, ocular Stevens Johnson Syndrome, ocular rosacea (with or without meibomian gland dysfunction)). The ocular disease, disorder, or condition does not include macular degeneration, such as AMD, or Stargardt's disease. In one illustration, in the ocular disease, disorder, or condition, the amount or concentration of MDA or HNE is increased in the ocular tissues or cells. For example, the amount or concentration of aldehydes (e.g., MDA or HNE) is increased for at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 5 fold, 10 fold as compared to that in normal ocular tissues or cells. Compounds described herein, such as Compound 9, decrease aldehyde (e.g., MDA and HNE) concentration in a time-dependent manner. The amount or concentration of aldehydes (e.g., MDA or HNE) can be measured by methods or techniques known in the art, such as those described in Tukozkan et al., Furat Tip Dergisi 11: 88-92 (2006).

In one class, the ocular disease, disorder, or condition is dry eye syndrome. In a second class, the ocular disease, disorder, or condition is a condition associated with PRK healing and other corneal healing. For example, the invention is directed to advancing PRK healing or other corneal healing, comprising administering to a subject in need thereof a compound described herein. In a third class, the ocular disease, disorder, or condition is an ocular condition associated with high aldehyde levels as a result of inflammation (e.g., uveitis, scleritis, ocular Stevens Johnson Syndrome, and ocular rosacea (with or without meibomian gland dysfunction). In a fourth class, the ocular disease, disorder, or condition is keratoconus, cataracts, bullous and other keratopathy, Fuchs' endothelial dystrophy, ocular cicatricial pemphigoid, or allergic conjunctivitis. The compound described herein may be administered topically or systemically, as described herein below.

In a second embodiment, the invention relates to the treatment, prevention, and/or reduction of a risk of a skin disorder or condition or a cosmetic indication, in which aldehyde toxicity is implicated in the pathogenesis, comprising administering to a subject in need thereof a compound described herein. The skin disorder or condition includes, but is not limited to, psoriasis, scleroderma, topical (discoid) lupus, contact dermatitis, atopic dermatitis, allergic dermatitis, radiation dermatitis, acne vulgaris, and Sjogren-Larsson Syndrome and other ichthyosis, and the cosmetic indication is solar elastosis/wrinkles, skin tone firmness, puffiness, eczema, smoke or irritant induced skin changes, dermal incision, or a skin condition associated burn and/or wound. In some embodiments, the invention related to age-related diseases, disorders, or conditions of the skin, as described herein.

Various skin disorders or conditions, such as atopic dermatitis, topical (discoid) lupus, psoriasis and scleroderma, are characterized by high MDA and HNE levels (Br J Dermatol 149: 248 (2003); JEADV 26: 833 (2012); Clin Rheumatol 25: 320 (2006)). In addition, ichthyosis characteristic of the Sjogren-Larsson Syndrome (SLS) originates from accumulation of fatty aldehydes, which disrupts the normal function and secretion of lamellar bodies (LB) and leads to intercellular lipid deposits in the Strateum Corneum (SC) and a defective water barrier in the skin layer (W. B. Rizzo et al. (2010)). The enzyme, fatty aldehyde dehydrogenase, that metabolizes aldehydes is dysfunctional in SLS patients. Thus, compounds that reduce or eliminate aldehydes, such as the compounds described herein, can be used to treat, prevent, and/or reduction of a risk of skin disorders or conditions in which aldehyde toxicity is implicated in the pathogenesis, such as those described herein. Furthermore, with an improvement to the water barrier and prevention of aldehyde-mediated inflammation (including fibrosis and elastosis (Chairpotto et al. (2005)), many cosmetic indications, such as solar elastosis/wrinkles, skin tone, firmness (puffiness), eczema, smoke or irritant induced skin changes and dermal incision cosmesis, and skin conditions associated with burn and/or wound can be treated using the method of the invention.

In one class, the skin disease, disorder, or condition is psoriasis, scleroderma, topical (discoid) lupus, contact dermatitis, atopic dermatitis, allergic dermatitis, radiation dermatitis, acne vulgaris, or Sjogren-Larsson Syndrome and other ichthyosis. In one exemplification, the skin disease, disorder, or condition is contact dermatitis, atopic dermatitis, allergic dermatitis, radiation dermatitis, or Sjogren-Larsson Syndrome and other ichthyosis. In a second class, the cosmetic indication is solar elastosis/wrinkles, skin tone firmness, puffiness, eczema, smoke or irritant induced skin changes, dermal incision, or a skin condition associated burn and/or wound.

In a third embodiment, the invention relates to the treatment, prevention, and/or reduction of a risk of a condition associated with the toxic effects of blister agents or burns from alkali agents in which aldehyde toxicity is implicated in the pathogenesis, comprising administering to a subject in need thereof a compound described herein.

Blister agents include, but are not limited to, sulfur mustard, nitrogen mustard, and phosgene oxime. Toxic or injurious effects of blister agents include pain, irritation, and/or tearing in the skin, eye, and/or mucous, and conjunctivitis and/or corneal damage to the eye. Sulfur mustard is the compound bis(2-chlorethyl) sulfide. Nitrogen mustard includes the compounds bis(2-chlorethyl)ethylamine, bis(2-chlorethyl)methylamine, and tris(2-chlorethyl)amine. Sulfur mustard or its analogs can cause an increase in oxidative stress and in particular in HNE levels, and by depleting the antioxidant defense system and thereby increasing lipid peroxidation, may induce an oxidative stress response and thus increase aldehyde levels (Jafari et al. (2010); Pal et al. (2009)). Antioxidants, such as Silibinin, when applied topically, attenuate skin injury induced from exposure to sulfur mustard or its analogs, and increased activities of antioxidant enzymes may be a compensatory response to reactive oxygen species generated by the sulfur mustard (Jafari et al. (2010); Tewari-Singh et al. (2012)). Further, intervention to reduce free radical species was an effective treatment post exposure for phosgene induced lung injury (Sciuto et al. (2004)). Thus, compounds that reduce or eliminate aldehydes, such as compounds described herein, can be used to treat, prevent, and/or reduce a risk of a condition associated with the toxic effects of blister agents, such as sulfur mustard, nitrogen mustard, and phosgene oxime.

Alkali agents include, but are not limited to, lime, lye, ammonia, and drain cleaners. Compounds that reduce or eliminate aldehydes, such as compounds described herein, can be used to treat, prevent, and/or reduce a risk of a condition associated with burns from an alkali agent.

In a fourth embodiment, the invention relates to the treatment, prevention, and/or reduction of a risk of an autoimmune, immune-mediated, inflammatory, cardiovascular, or neurological disease, disorder, or condition, or metabolic syndrome, or diabetes, in which aldehyde toxicity is implicated in the pathogenesis, comprising administering to a subject in need thereof a compound described herein. The autoimmune or immune-mediated disease, disorder, or condition includes, but is not limited to, lupus, scleroderma, asthma, chronic obstructive pulmonary disease (COPD), and rheumatoid arthritis. The inflammatory disease, disorder, or condition includes, but is not limited to, rheumatoid arthritis, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), sepsis, and fibrosis (e.g., renal, hepatic, pulmonary, and cardiac fibrosis). The cardiovascular disease, disorder, or condition includes, but is not limited to, atherosclerosis and ischemic-reperfusion injury. The neurological disease, disorder, or condition includes, but is not limited to, Parkinson's disease, Alzheimer's disease, succinic semialdehyde dehydrogenase deficiency, multiple sclerosis, amyotrophic lateral sclerosis, and the neurological aspects of Sjogren-Larsson Syndrome (cognitive delay and spasticity).

A skilled person would understand that the disease, disorder, or condition listed herein may involve more than one pathological mechanism. For example, a disease, disorder, or condition listed herein may involve dysregulation in the immunological response and inflammatory response. Thus, the above categorization of a disease, disorder, or condition is not absolute, and the disease, disorder, or condition may be considered an immunological, an inflammatory, a cardiovascular, a neurological, and/or metabolic disease, disorder, or condition.

Individuals with deficiencies in aldehyde dehydrogenase are found to have high aldehyde levels and increased risk of Parkinson's disease (PNAS 110:636 (2013)) and Alzheimer's disease (BioChem Biophys Res Commun. 273:192 (2000)). In Parkinson's disease, aldehydes specifically interfere with dopamine physiology (Free Radic Biol Med, 51: 1302 (2011); Mol Aspects Med, 24: 293 (2003); Brain Res, 1145: 150 (2007)). In addition, aldehydes levels are elevated in multiple sclerosis, amyotrophic lateral sclerosis, autoimmune diseases such as lupus, rheumatoid arthritis, lupus, psoriasis, scleroderma, and fibrotic diseases, and increased levels of HNE and MDA are implicated in the progression of atherosclerosis and diabetes (J. Cell. Mol. Med., 15: 1339 (2011); Arthritis Rheum 62: 2064 (2010); Clin Exp Immunol, 101: 233 (1995); Int J Rheum Dis, 14: 325 (2011); JEADV 26: 833 (2012); Clin Rheumatol 25: 320 (2006); Gut 54: 987 (2005); J Am Soc Nephrol 20: 2119 (2009)). MDA is further implicated in the increased formation of foam cells leading to atherosclerosis (Leibundgut et al., Current Opinion in Pharmacology 13: 168 (2013)). Also, aldehyde-related toxicity plays an important role in the pathogenesis of many inflammatory lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD) (Bartoli et al., Mediators of Inflammation 2011, Article 891752). Thus, compounds that reduce or eliminate aldehydes, such as compounds described herein, can be used to treat, prevent, and/or reduce a risk of an autoimmune, immune-mediated, inflammatory, cardiovascular, or neurological disease, disorder, or condition, or metabolic syndrome, or diabetes. For example, compounds described herein prevent aldehyde-mediated cell death in neurons. Further, compounds described herein downregulate a broad spectrum of pro-inflammatory cytokines and/or upregulate anti-inflammatory cytokines, which indicates that compounds described herein are useful in treating inflammatory diseases, such as multiple sclerosis and amyotrophic lateral sclerosis.

As discussed above, a disclosed composition may be administered to a subject in order to treat or prevent macular degeneration and other forms of retinal disease whose etiology involves the accumulation of A2E and/or lipofuscin. Other diseases, disorders, or conditions characterized by the accumulation A2E may be similarly treated.

In one embodiment, a compound is administered to a subject that reduces the formation of A2E. For example, the compound may compete with PE for reaction with trans-RAL, thereby reducing the amount of A2E formed. In another embodiment, a compound is administered to a subject that prevents the accumulation of A2E. For example, the compound competes so successfully with PE for reaction with trans-RAL, no A2E is formed.

Individuals to be treated fall into three groups: (1) those who are clinically diagnosed with macular degeneration or other forms of retinal disease whose etiology involves the accumulation of A2E and/or lipofuscin on the basis of visual deficits (including but not limited to dark adaptation, contrast sensitivity and acuity) as determined by visual examination and/or electroretinography, and/or retinal health as indicated by fundoscopic examination of retinal and RPE tissue for drusen accumulations, tissue atrophy and/or lipofuscin fluorescence; (2) those who are pre-symptomatic for macular degenerative disease but thought to be at risk based on abnormal results in any or all of the same measures; and (3) those who are pre-symptomatic but thought to be at risk genetically based on family history of macular degenerative disease and/or genotyping results showing one or more alleles or polymorphisms associated with the disease. The compositions are administered topically or systemically at one or more times per month, week or day. Dosages may be selected to avoid side effects, if any, on visual performance in dark adaptation. Treatment is continued for a period of at least one, three, six, or twelve or more months. Patients may be tested at one, three, six, or twelve months or longer intervals to assess safety and efficacy. Efficacy is measured by examination of visual performance and retinal health as described above.

In one embodiment, a subject is diagnosed as having symptoms of macular degeneration, and then a disclosed compound is administered. In another embodiment, a subject may be identified as being at risk for developing macular degeneration (risk factors include a history of smoking, age, female gender, and family history), and then a disclosed compound is administered. In another embodiment, a subject may have dry AMD in both eye, and then a disclosed compound is administered. In another embodiment, a subject may have wet AMD in one eye but dry AMD in the other eye, and then a disclosed compound is administered. In yet another embodiment, a subject may be diagnosed as having Stargardt disease and then a disclosed compound is administered. In another embodiment, a subject is diagnosed as having symptoms of other forms of retinal disease whose etiology involves the accumulation of A2E and/or lipofuscin, and then the compound is administered. In another embodiment a subject may be identified as being at risk for developing other forms of retinal disease whose etiology involves the accumulation of A2E and/or lipofuscin, and then the disclosed compound is administered. In some embodiments, a compound is administered prophylactically. In some embodiments, a subject has been diagnosed as having the disease before retinal damage is apparent. For example, a subject is found to carry a gene mutation for ABCA4 and is diagnosed as being at risk for Stargardt disease before any ophthalmologic signs are manifest, or a subject is found to have early macular changes indicative of macular degeneration before the subject is aware of any effect on vision. In some embodiments, a human subject may know that he or she is in need of the macular generation treatment or prevention.

In some embodiments, a subject may be monitored for the extent of macular degeneration. A subject may be monitored in a variety of ways, such as by eye examination, dilated eye examination, fundoscopic examination, visual acuity test, and/or biopsy. Monitoring can be performed at a variety of times. For example, a subject may be monitored after a compound is administered. The monitoring can occur, for example, one day, one week, two weeks, one month, two months, six months, one year, two years, five years, or any other time period after the first administration of a compound. A subject can be repeatedly monitored. In some embodiments, the dose of a compound may be altered in response to monitoring.

In some embodiments, the disclosed methods may be combined with other methods for treating or preventing macular degeneration or other forms of retinal disease whose etiology involves the accumulation of A2E and/or lipofuscin, such as photodynamic therapy. For example, a patient may be treated with more than one therapy for one or more diseases or disorders. For example, a patient may have one eye afflicted with dry form AMD, which is treated with a compound of the invention, and the other eye afflicted with wet form AMD which is treated with, e.g., photodynamic therapy.

In some embodiments, a compound for treating or preventing macular degeneration or other forms of retinal disease whose etiology involves the accumulation of A2E and/or lipofuscin may be administered chronically. The compound may be administered daily, more than once daily, twice a week, three times a week, weekly, biweekly, monthly, bimonthly, semiannually, annually, and/or biannually.

Sphingosine 1-phosphate, a bioactive signaling molecule with diverse cellular functions, is irreversibly degraded by the endoplasmic reticulum enzyme sphingosine 1-phosphate lyase, generating trans-2-hexadecenal and phosphoethanolamine. It has been demonstrated that trans-2-hexadecenal causes cytoskeletal reorganization, detachment, and apoptosis in multiple cell types via a JNK-dependent pathway. See Biochem Biophys Res Commun. 2012 Jul. 20; 424(1):18-21. These findings and the known chemistry of related α,β-unsaturated aldehydes raise the possibility that trans-2-hexadecenal interact with additional cellular components. It was shown that it reacts readily with deoxyguanosine and DNA to produce the diastereomeric cyclic 1,N(2)-deoxyguanosine adducts 3-(2-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8R-hydroxy-6R-tridecylpyrimido[1,2-a]purine-10(3H)one and 3-(2-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8S-hydroxy-6S-tridecylpyrimido[1,2-a]purine-10(3H)one. These findings demonstrate that trans-2-hexadecenal produced endogenously by sphingosine 1-phosphate lyase react directly with DNA forming aldehyde-derived DNA adducts with potentially mutagenic consequences.

Succinic semialdehyde dehydrogenase deficiency (SSADHD), also known as 4-hydroxybutyric aciduria or gamma-hydroxybutyric aciduria, is the most prevalent autosomal-recessively inherited disorder of GABA metabolism (Vogel et al. 2013), manifests a phenotype of developmental delay and hypotonia in early childhood, and severe expressive language impairment and obsessive-compulsive disorder in adolescence and adulthood. Epilepsy occurs in half of patients, usually as generalized tonic-clonic seizures although sometimes absence and myoclonic seizures occur (Pearl et al. 2014). Greater than two-thirds of patients manifest neuropsychiatric problems (i.e., ADHD, OCD and aggression) in adolescence and adulthood, which can be disabling. Metabolically, there is accumulation of the major inhibitory neurotransmitter GABA and gamma-hydroxybutyrate (GHB), a neuromodulatory monocarboxylic acid (Snead and Gibson 2005). In addition, several other intermediates specific to this disorder have been detected both in patients and the corresponding murine model. Vigabatrin (VGB; γ-vinylGABA), an irreversible inhibitor of GABA-transaminase, is a logical choice for treatment of SSADH deficiency because it will prevent the conversion of GABA to GHB. Outcomes have been mixed, and in selected patients treatment has led to deterioration (Good 2011; Pellock 2011; Escalera et al. 2010; Casarano et al. 2011; Matern et al. 1996; Al-Essa et al. 2000). Targeted therapy for SSADH deficiency remains elusive and interventions palliative.

5. Pharmaceutically Acceptable Compositions

The compounds and compositions, according to the method of the present invention, are administered using any amount and any route of administration effective for treating or lessening the severity of a disorder provided above. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular agent, its mode of administration, and the like. Compounds of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression “dosage unit form” as used herein refers to a physically discrete unit of agent appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific effective dose level for any particular patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed, and like factors well known in the medical arts.

Pharmaceutically acceptable compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), buccally, as an oral or nasal spray, or the like, depending on the severity of the infection being treated. In certain embodiments, the compounds of the invention are administered orally or parenterally at dosage levels of about 0.01 mg/kg to about 50 mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of a compound of the present invention, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this invention. Additionally, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

The compounds of the invention can also be administered topically, such as directly to the eye, e.g., as an eye-drop or ophthalmic ointment. Eye drops typically comprise an effective amount of at least one compound of the invention and a carrier capable of being safely applied to an eye. For example, the eye drops are in the form of an isotonic solution, and the pH of the solution is adjusted so that there is no irritation of the eye. In many instances, the epithelial barrier interferes with penetration of molecules into the eye. Thus, most currently used ophthalmic drugs are supplemented with some form of penetration enhancer. These penetration enhancers work by loosening the tight junctions of the most superior epithelial cells (Burstein, 1985, Trans Ophthalmol Soc U K 104(Pt 4): 402-9; Ashton et al., 1991, J Pharmacol Exp Ther 259(2): 719-24; Green et al., 1971, Am J Ophthalmol 72(5): 897-905). The most commonly used penetration enhancer is benzalkonium chloride (Tang et al., 1994, J Pharm Sci 83(1): 85-90; Burstein et al, 1980, Invest Ophthalmol Vis Sci 19(3): 308-13), which also works as preservative against microbial contamination. It is typically added to a final concentration of 0.01-0.05%.

The term “biological sample”, as used herein, includes, without limitation, cell cultures or extracts thereof; biopsied material obtained from a mammal or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.

All features of each of the aspects of the invention apply to all other aspects mutatis mutandis.

In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.

EXEMPLIFICATION

As depicted in the Examples below, in certain exemplary embodiments, compounds are prepared according to the following general procedures. It will be appreciated that, although the general methods depict the synthesis of certain compounds of the present invention, the following general methods, and other methods known to one of ordinary skill in the art, can be applied to all compounds and subclasses and species of each of these compounds, as described herein.

Example 1: General Reaction Sequence for Compounds

Deuterium-labeled aldehyde trapping agents were made as described in U.S. patent application publication US 2013/0190500, published Jul. 23, 2013, optionally using deuterium-labeled intermediates at the sites indicated in Scheme 1. Exemplary methods are described further below.

Example 2: Synthesis of A-1

1-(3-ethoxy-2,3-dioxopropyl)pyridin-1-ium bromide. To a 2 L round bottom flask was charged ethanol (220 mL) and pyridine (31 g, 392 mmol), and the resulting solution was stirred at a moderate rate of agitation under nitrogen. To this solution was added ethyl bromopyruvate (76.6 g, 354 mmol) in a slow, steady stream. The reaction mixture was allowed to stir at 65±5° C. for 2 hours.

Example 3: Synthesis of A-2a

1-(6-chloro-2-(ethoxycarbonyl)quinolin-3-yl)pyridin-1-ium bromide. Upon completion of the 2 hour stir time in Example 2, the reaction mixture was slowly cooled to 18-22° C. The flask was vacuum-purged three times at which time 2-amino-5-chloro-benzaldehyde (ACB) (50.0 g, 321 mmol) was added directly to the reaction flask as a solid using a long plastic funnel. Pyridine (64.0 g, 809 mmol) was added followed by an EtOH rinse (10 mL) and the reaction mixture was heated at 80±3° C. under nitrogen for about 16 hours (overnight) at which time HPLC analysis indicated that the reaction was effectively complete.

Example 4: Synthesis of A-2b

1-(6-chloro-2-(ethoxycarbonyl)quinolin-3-yl-4-d)pyridin-1-ium. Compound A-2b is prepared in a manner similar to A-2a (See Example 3), substituting 2-amino-5-chloro-benzaldehyde (ACB) for 2-amino-5-chloro-benzaldehyde-d.

Example 5: Synthesis of A-3a

Ethyl 3-amino-6-chloroquinoline-2-carboxylate. The reaction mixture from Example 3 was cooled to about 70° C. and morpholine (76.0 g, 873 mmol)) was added to the 2 L reaction flask using an addition funnel. The reaction mixture was heated at 80±2° C. for about 2.5 hours at which time the reaction was considered complete by HPLC analysis (area % of A-3a stops increasing). The reaction mixture was cooled to 10-15° C. for the quench, work up, and isolation.

To the 2 L reaction flask was charged water (600 g) using the addition funnel over 30-60 minutes, keeping the temperature below 15° C. by adjusting the rate of addition and using a cooling bath. The reaction mixture was stirred for an additional 45 minutes at 10-15° C. then the crude A-3a was isolated by filtration using a Buchner funnel. The cake was washed with water (100 mL×4) each time allowing the water to percolate through the cake before applying a vacuum. The cake was air dried to provide crude A-3a as a nearly dry brown solid. The cake was returned to the 2 L reaction flask and heptane (350 mL) and EtOH (170 mL) were added, and the mixture heated to 70±3° C. for 30-60 minutes. The slurry was cooled to 0-5° C. and isolated by filtration under vacuum. The A-3a was dried in a vacuum drying oven under vacuum and 35±3° C. overnight (16-18 hours) to provide A-3a as a dark green solid.

Example 6: Synthesis of A-3b

Ethyl 3-amino-6-chloroquinoline-2-carboxylate-4-d. Compound A3-b is prepared in a similar manner as compound A3-a (See Example 5), substituting the reaction mixture of A2-a for that of A2-b.

Example 7: Synthesis of NS2

2-(3-amino-6-chloroquinolin-2-yl)propan-2-ol. To a 2 L round bottom flask was charged methylmagnesium chloride (200 mL of 3.0 M solution in THF, 600 mmol). The solution was cooled to 0-5° C. using an ice bath.

A 500 mL flask (magnetic stirring) was charged with 22.8 grams A-3a from Example 5 and THF (365 mL), stirred to dissolve, and then transferred to an addition funnel on the 2 L Reaction Flask. The A-3a solution was added drop-wise to the reaction flask over 5.75 hours, keeping the temperature of the reaction flask between 0-5° C. throughout the addition. At the end of the addition the contents of the flask were stirred for an additional 15 minutes at 0-5° C. then the cooling bath was removed and the reaction was allowed to stir overnight at ambient temperature.

The flask was cooled in an ice bath and the reaction mixture was carefully quenched by adding EtOH (39.5 g, 857 mmol) drop-wise to the reaction mixture, keeping the temperature of the reaction mixture below 15° C. during the course of the addition. An aqueous solution of NH₄Cl (84.7 g NH₄Cl in 415 mL water) was then carefully added and the mixture stirred under moderate agitation for about 30 minutes then transferred to a separatory funnel to allow the layers to separate. Solids were present in the aqueous phase so HOAc (12.5 g) was added and the contents swirled gently to obtain a nearly homogeneous lower aqueous phase. The lower aqueous layer was transferred back to the 2 L reaction flask and stirred under moderate agitation with 2-methylTHF (50 mL) for about 15 minutes. The original upper organic layer was reduced in volume to approximately 40 mL using a rotary evaporator at ≤40° C. and vacuum as needed. The phases in the separatory funnel were separated and the upper 2-MeTHF phase combined with the product residue, transferred to a 500 mL flask, and vacuum distilled to an approximate volume of 25 mL. To this residue was added 2-MeTHF (50 mL) and distilled to an approximate volume of 50 mL. The crude compound NS2 solution was diluted with 2-MeTHF (125 mL), cooled to 5-10° C., and 2M H₂SO₄ (aq) (250 mL) was slowly added and the mixture stirred for 30 minutes as the temperature was allowed to return to ambient. Heptane (40 mL) was charged and the reaction mixture stirred for an additional 15 minutes then transferred to a separatory funnel, and the layers were allowed to separate. The lower aqueous product layer was extracted with additional heptane (35 mL), then the lower aqueous phase was transferred to a 1 L reaction flask equipped with a mechanical stirrer, and the mixture was cooled to 5-10° C. The combined organic layers were discarded. A solution of 25% NaOH (aq) was prepared (NaOH, 47 g, water, 200 mL) and slowly added to the 1 L reaction flask to bring the pH to a range of 6.5-8.5.

EtOAc (250 mL) was added and the mixture was stirred overnight. The mixture was transferred to a separatory funnel and the lower phase discarded. The upper organic layer was washed with brine (25 mL), then the upper organic product layer was reduced in volume on a rotary evaporator to obtain a obtain the crude compound NS2 as a dark oil that solidified within a few minutes. The crude compound NS2 was dissolved in EtOAc (20 mL) and filtered through a plug of silica gel (23 g) eluting with 3/1 heptane/EtOAc until all compound NS2 was eluted (approximately 420 mL required) to remove most of the dark color of compound NS2. The solvent was removed in vacuo to provide 14.7 g of compound NS2 as a tan solid. Compound NS2 was taken up in EtOAc (25 mL) and eluted through a column of silica gel (72 g) using a mobile phase gradient of 7/1 heptane/EtOAc to 3/1heptane/EtOAc (1400 mL total). The solvent fractions containing compound NS2 were stripped. Compound NS2 was diluted with EtOAc (120 mL) and stirred in a flask with Darco G-60 decolorizing carbon (4.0 g) for about 1 hour. The mixture was filtered through celite using a fritted funnel, rinsing the cake with EtOAc (3×15 mL). The combined filtrates were stripped on a rotary evaporator and compound NS2 dissolved in heptane (160 mL)/EtOAc (16 mL) at 76° C. The homogeneous solution was slowly cooled to 0-5° C., held for 2 hours, then compound NS2 was isolated by filtration. After drying in a vacuum oven for 5 hours at 35° C. under best vacuum, compound NS2 was obtained as a white solid. HPLC purity: 100% (AUC); HPLC (using standard conditions): A-2: 7.2 minutes; A-3: 11.6 minutes.

Example 8: Synthesis of I-1

2-(6-chloroquinolin-2-yl)propan-1,1,1,3,3,3-d₆-2-ol. Compound I-1 was prepared in a similar manner to compound NS2 (See Example 7), substituting methylmagnesium chloride with methyl-d₃-magnesium iodide (99 atom % D). The reaction of A3-a with 5.3 mol equiv 1.0 M methyl-d₃-magnesium iodide (99 atom % D) in ether/THF gave a 20% yield of I-1. MS (ESI): m/z 242.9 (M+1); ¹H NMR (CDCl₃, 300 MHz) δ: 7.80 (d, J=6 Hz, 1H), 7.51 (d, J=2 Hz, 1H), 7.33 (dd, J=2 and 2 Hz, 1H), 7.07 (s, 1H), 4.68 (br s, 2H), 3.83 (br s, 1H); ¹³C NMR (CDCl₃, 75 MHz) δ: 154.3, 139.5, 139.1, 132.4, 130.6, 130.0, 126.3, 123.7, 116.4, 76.8, 74.9.

Example 9: Synthesis of I-2

2-(6-chloroquinolin-2-yl-4-d)propan-2-ol. Compound 1-2 is prepared in a similar manner to compound NS2 (See Example 7), substituting A3-a for A3-b.

Preparation of ACB

After a N₂ atmosphere had been established and a slight stream of N₂ was flowing through the vessel, platinum, sulfided, 5 wt % on carbon, reduced, dry (9.04 g, 3.0 wt % vs the nitro substrate) was added to a 5 L heavy walled pressure vessel equipped with a large magnetic stir-bar and a thermocouple. MeOH (1.50 L), 5-chloro-2-nitrobenzaldehyde (302.1 g, 1.63 mol), further MeOH (1.50 L) and Na₂CO₃ (2.42 g, 22.8 mmol, 0.014 equiv) were added. The flask was sealed and stirring was initiated at 450 rpm. The solution was evacuated and repressurized with N₂ (35 psi), 2×. The flask was evacuated and repressurized with H₂ to 35 psi. The temperature of the solution reached 30° C. w/in 20 min. The solution was then cooled with a water bath. Ice was added to the water bath to maintain a temperature below 35° C. Every 2 h, the reaction was monitored by evacuating and repressurizing with N₂ (5 psi), 2× prior to opening. The progress of the reaction could be followed by TLC: 5-Chloro-2-nitrobenzaldehyde (R_(f)=0.60, CH₂Cl₂, UV) and the intermediates (R_(f)=0.51, CH₂Cl₂, UV and R_(f)=0.14, CH₂Cl₂, UV) were consumed to give ACB (R_(f)=0.43, CH₂Cl₂, UV). At 5 h, the reaction had gone to 98% completion (GC), and was considered complete. To a 3 L medium fritted funnel was added celite (ca. 80 g). This was settled with MeOH (ca. 200 mL) and pulled dry with vacuum. The reduced solution was transferred via cannula into the funnel while gentle vacuum was used to pull the solution through the celite plug. This was chased with MeOH (150 mL 4×). The solution was transferred to a 5 L three-necked round-bottom flask. At 30° C. on a rotavap, solvent (ca. 2 L) was removed under reduced pressure. An N₂ blanket was applied. The solution was transferred to a 5 L four-necked round-bottomed flask equipped with mechanical stirring and an addition funnel. Water (2.5 L) was added dropwise into the vigorously stirring solution over 4 h. The slurry was filtered with a minimal amount of vacuum. The collected solid was washed with water (1.5 L 2×), 2-propanol (160 mL) then hexanes (450 mL 2×). The collected solid (a canary yellow, granular solid) was transferred to a 150×75 recrystallizing dish. The solid was then dried under reduced pressure (26-28 in Hg) at 40° C. overnight in a vacuum-oven. ACB (>99A % by HPLC) was stored under a N₂ atmosphere at 5° C.

Example 10: In Vitro Assays

LDH Cytotoxicity Assay

Primary rat cortical cultures are placed in an incubator for 24 or 48 hours and treated with various concentrations of disclosed compounds. Then 20 μL of the culture media is removed for an LDH assay as described in Bergmeyer et al., Methods of Enzymatic Analysis, 3^(rd) ed. (1983).

ELISA Assay to Determine Amount of Circulating Cytokines

Male C57BI/6 mice are dosed with disclosed compounds 30 minutes before they were exposed to LPS (20 mg/kg). Two hours after the LPS exposure, blood is collected from the mice and an ELISA is conducted to determine the amount of circulating cytokines. Treatment with disclosed compounds leads to reduction in proinflammatory cytokines, such as IL-5 and IL-1β, IL-17, and TNF. Also, treatment with disclosed compounds results in elevated anti-inflammatory cytokines, such as IL-10. In addition, various other chemokines, such as eotaxin, IL-12, IP-10, LIF, MCP-1, MIG, MIP, and RANTES, are also decreased by treatment with disclosed compounds.

Assay to Evaluate Efficacy in Treating Contact Dermatitis

To determine the efficacy of the disclosed compounds in treating contact dermatitis, phorbol myristate acetate (“PMA”) is applied topically (2.5 μg in 20 μL) to both the anterior and posterior portions of the right pinna of mice (N=10 per group). As a control, the left pinna receives 20 μL of ethanol (PMA excipient) to both the anterior and posterior portions. Six hours after the PMA application, both the right and left pinna thickness is determined. Measurements are determined at least twice from the same region of both ears, with care taken not to include hair or folded pinna.

Assay to Evaluate the Efficacy in Treating Allergic Dermatitis

To measure the efficacy of the disclosed compounds in treating allergic dermatitis, oxazolone (“OXL”) is applied (1.5%, 100 μL in acetone) to the shaved abdomens of mice. Seven days later, the thickness of the pinna of the OXL treated mice is determined. Then the disclosed compounds (100 mg/kg) or the vehicle (i.e., Captisol) is administered intraperitoneally to mice followed by topical application of OXL (1%, 20 μL) 30 min later to both the anterior and posterior portions of the right pinna. As a control, the left pinna receives 20 μL of acetone (OXL excipient) to both the anterior and posterior portions. The thickness of the pinna of both ears is measured again 24 hours later. N=10 per group.

Assay to Measure Aldehyde Trapping

To separate reaction vials is added each disclosed compound, (0.064 mmol), MDA salt (22.7% MDA, 0.064 mmol), and glyceryl trioleate (600 mg). To the mixture is added 20 wt % Capitsol in aqueous PBS (˜2.5 ml), followed by linoleic acid (600 mg). The reaction mixture is stirred vigorously at ambient temperature and monitored by LC/MS. The disclosed compounds quickly react with MDA to form MDA adducts.

Schiff Base Confirmation

UV/VIS spectroscopy is used to monitor Schiff base condensation of RAL with the primary amine of a compound of the invention. The in vitro analysis of the Schiff base condensation product with RAL is performed for the disclosed compounds.

In the solution phase analysis, the λ_(max) value of both the free compound and the RAL Schiff base condensation product (RAL-SBC) are measured along with the value for tau of the RAL-SBC. As used herein, “RAL-SBC” means the Schiff base condensation product of RAL and a RAL-compound. Solution phase analysis is performed using a 100:1 mixture of compound and RAL using protocols known in the art. Several solvent systems were tested including aqueous, ethanol, octanol, and chloroform:methanol (various e.g., 2:1). The solution kinetics are measured and found to be highly dependent on solvent conditions.

Solid phase analysis of the Schiff base condensation is also performed using a 1:1 mixture of compound to RAL. The solid phase analysis is performed using protocols known in the art. The mixture is dried under nitrogen and condensation reaction occurs to completion.

Lipid phase analysis is performed using protocols known in the art and λ_(max), tau (RAL-SBC vs. APE/A2PE), and competitive inhibition are measured. Liposome conditions are closer to in situ conditions.

ERG Analysis of Dark Adaptation

Dark adaptation is the recovery of visual sensitivity following exposure to light. Dark adaptation has multiple components including both fast (neuronal) processes and a slow (photochemical) process. Regeneration of visual pigment is related to the slow photochemical process. Dark adaptation rates are measured for several reasons. Night blindness results from a failure to dark adapt (loss of visual light sensitivity). It is possible to find a safe dose for night vision by measuring drug effects on dark adapted visual light sensitivity.

An electroretinogram (ERG) is used to measure dark adaptation under normal vs. drug conditions. ERG is the measurement of the electric field potential emitted by retinal neurons during their response to an experimentally defined light stimulus. More specifically, ERG measures retinal field potentials at the cornea after a flash of light (e.g., 50 ms). Field strengths are 102 to 103 microvolts, originating in retinal cells.

ERG is a non-invasive measurement which can be performed on either living subjects (human or animal) or a hemisected eye in solution that has been removed surgically from a living animal. ERG requires general anesthesia which slows dark adaptation and must be factored into experimental design.

In a typical ERG analysis of dark adaptation experiment, every rat is dark adapted for hours to reach a consistent state of light sensitivity. The rat is then “photo-bleached,” i.e., exposed briefly to light strong enough to transiently deplete the retina of free 11-cis-RAL (e.g., 2 min at 300 lux). The rat is then returned to dark immediately to initiate dark adaptation, i.e., recovery of light sensitivity due to regeneration of visual pigment. ERG is used to measure how quickly the rat adapts to dark and recovers light sensitivity. Specifically, a criterion response variable is defined for light sensitivity.

The ERG measurement is taken after a specific duration of post-bleach dark recovery (e.g., 30 min) determined previously by kinetic analysis. A curve fit is used to calculate value for the sensitivity variable and shows recovery with anesthesia in the same rat including dark adaptation kinetics for Y₅₀ and σ. Slower adaptation is observed with less light sensitivity where Y₅₀ reaches −4.0 and tau=22.6 min. Faster adaptation is observed with more light sensitivity where Y₅₀ reaches −5.5 and tau=9.2 min.

The same paradigm as described above is followed for dose ranging. In the ERG dose ranging protocol, compounds i.p. lowers light sensitivity of dark adapted rats in a dose dependent manner. The effect on vision decreases after 3 hours.

NMR Analysis of RAL Reaction

NMR spectroscopy is used to monitor Schiff base condensation and ring formation of RAL with the primary amine of a compound of the invention.

Inhibition of A2E Formation

This experiment is designed to establish proof of concept that chronic i.p. injection of a RAL-trap compound lowers the accumulation rate of A2E in wild type Sprague Dawley rats. These experiments compare the treatment efficacy of RAL-trap compounds to that of control compounds and lack of treatment.

Materials and Methods:

The study is performed with wild type Sprague Dawley rats. Rat treatment groups include, for example, 8 rats of mixed gender per treatment condition. Each animal is treated with one of the following conditions:

-   -   Controls: (1) 13-cis retinoic acid to inhibit retinoid binding         sites of visual cycle proteins as a protocol control, in that         such treatment reduces the amount of free trans-RAL that is         released and thereby available to form A2E, but with undesirable         side effects of night blindness, and (2) a commercially         available compound known clinically to modulate retinal function         in humans and known experimentally to form a Schiff base adduct         with free RAL, both in vitro and in vivo in animal models.     -   Vehicle     -   Compound     -   Untreated

The disclosed compounds are tested across a dose range including 1, 5, 15, and 50 mg/kg. Treatment is administered daily for 8 weeks by i.p. injection.

Chemistry:

The experiments use a variety of chemistry services. For example, these experiments use commercially available compounds with analytical specification sheets to characterize the impurities. Compounds are also synthesized. Compounds are prepared in quantities sufficient for the required dosing. Formulations of the compound are suitable for use in initial animal safety studies involving intraperitoneal (i.p.) injection. The following three attributes of the Schiff base reaction product of trans-RAL with compounds of the invention are determined:

-   -   stability with respect to reaction rates     -   absorption properties, specifically uv-vis absorption maxima and         extinction coefficients (see e.g., FIG. 5 in Rapp and Basinger,         Vision Res. 22:1097, 1982) or NMR spectral analysis of reaction         kinetics     -   log P and log D solubility values e.g. calculated         Biology and Biochemistry:

The experiments described herein use a variety of biology and biochemistry services. A “no effect level” (NOEL) dose of compounds of the invention for daily treatment with an eye drop formation is established, e.g., in the rabbit with an ocular irritation protocol and in the rodent with ERG measurement of dark adaptation in visual responses to light stimulation. After treatment and before eye enucleation, the following non-invasive assays are performed in animals, e.g., rabbits:

-   -   RPE and photoreceptor cell degeneration, as evident by fundus         photography (Karan, et al. 2005, PNAS 102:4164)     -   Extracellular drusen and intracellular lipofuscin as measured by         fundus fluorescent photography (Karan et al. 2005)

Light responses are characterized by ERG (Weng, et al., Cell 98:13, 1999). Intracellular A2E concentration of retinal RPE cell extracts is measured in all treated animals upon the conclusion of the treatment protocol using an analytical method such as those described by Karan et al., 2005; Radu et al., 2003; and Parish et al., PNAS 95:14609, 1998. For example, in a sample of treated animals, one eye is assayed, and the other eye is saved for histology analysis (as described below). In the remaining animals, both eyes are assayed separately for A2E formation.

In the post-treatment eyes set aside for histology (as described above), the morphology of retinal and RPE tissue is assessed with light microscopy histology techniques (Karan et al. 2005, with the exception that electron microscopy is not used in the experiments described herein).

The safety of the treatment regimen is assessed for example using a combination of:

-   -   Daily documented observation of animal behavior and feeding         habits throughout the treatment period     -   Visual performance as measured by ERG at the end of the         treatment period     -   Ocular histology at the end of the treatment

Example 11: Cross-Species Metabolite Profiling Test of Deuterated NS2 (NS2-D6; Compound I-1)

Purpose:

Once administered to an animal, small molecules may undergo a variety of reactions to produce an array of metabolites. The exact metabolites produced depend on many factors such as the animal, the molecular structure, and the tissue distribution of the molecule. Metabolism of small molecules may serve the purpose of increasing water solubility to aid in excretion of the molecule in the urine or feces, or may simply be the result of adventitious enzyme-catalyzed reactions. Exemplary metabolites include oxidation and glucuronidation products. It is often impossible to predict the distribution and amount of metabolites produced. One purpose of this study was to conduct a cross species metabolite profiling of test article NS2 in cryopreserved primary hepatocytes. As described below, a variety of NS2 metabolites were produced and the distribution of metabolites varied significantly across different species. It is often desirable to decrease the number and amount of metabolites of small molecule drugs, for example to increase drug half-life in the body and/or prevent conversion to toxic or inactive metabolites. With this knowledge in hand, deuteration of NS2 was explored as a possible route to decrease the number and amount of metabolites. Deuteration of a molecule (i.e., replacement of one or more hydrogen atoms with deuterium) often has significant effects on the rates of production of metabolites; however, the effects of deuteration on a given molecule are almost impossible to predict in most cases. Therefore, metabolite profiling was performed for deuterated-NS2 in human hepatocytes.

Study Conditions:

This study was performed under non-GLP conditions. All work was performed with appropriate local health regulations and ethical approval.

Experimental Design:

Sample Analysis

Samples were analyzed by LC-MS/MS using a SCIEX QTrap 5500 mass spectrometer coupled with an Agilent 1290 HPLC Infinity series, a CTC PAL chilled autosampler, all controlled by Analyst software. After separation on a C18 reverse phase HPLC column (Acquity UPLC HSS T3, 1.8, 2.1×50 mm) using an acetonitrile-water gradient system, peaks were analyzed by mass spectrometry (MS) using ESI ionization in Q1 scan mode. LC conditions are shown in Table 3 below.

TABLE 3 LC Gradient Time Flow rate % A % B (min) (mL/min) Mobile Phase Mobile Phase 0.05 0.6 100 2 5.0 0.6 60 40 6.0 0.6 5 95 6.4 0.6 5 95 6.41 0.6 100 0 6.8 0.6 100 0

Table 4 shows experimental parameters for metabolite profiling.

TABLE 4 Metabolite Profiling in Hepatocytes: Experimental Conditions Test Test Hepatocyte Cell Time Points Analytical Article conc. source count Profiled method NS2 (all 4 species) 3 μM Rat, dog, 1 × 10⁶ viable 0 and 120 min LC-MS/MS D-NS2 (human only) monkey (cyno) cells/mL and human Experimental Procedure

The test article was incubated in duplicate with primary, cryopreserved hepatocytes at 37° C. The cells were thawed, viable cells counted, and equilibrated according to the supplier's directions. After 30 min equilibration at 37° C. with gentle agitation, the test compound was added into the cells to give the desired final concentration of 3 μM. The cell suspension was incubated at 37° C. as described above. At the indicated times, samples were removed and mixed with an equal volume of ice-cold stop solution (methanol).

In parallel, a blank hepatocyte sample in the absence of test agent was incubated for 120 min and was used as a control to show the presence of peaks derived from the hepatocytes. Stopped reactions were incubated at least ten minutes on ice, and an additional volume of water was added. The samples are centrifuged to remove precipitated protein, and the supernatants were analyzed by LC-MS/MS.

A full scan mass spectrum (100-800 m/z) in both positive and negative ionization modes were run across the gradient to look for the presence of potential metabolites (novel masses and known Phase I and II metabolites such as: oxidation, sulfation, di-oxidation, dehydrogenation, sulfation+oxidation, glucuronidation, oxidation+glucuronidation, and glutathione conjugation). The mass spectrometry method is shown below in Table 5.

TABLE 5 Mass Spectrometry Method Development Test ESI Full Scan Article MW Polarization Mass Range NS2 236.7 Positive 100-800 CoteRX micronized Lot 093 (Origin Lot BR-NS2-11-01) D-NS2 242.7 Positive 100-800 (Lot 1509342002) D-NS2: deuterated N32 (C12H7ClN2Od6) Results

It was surprisingly found that deuteration of NS2 greatly reduced the number and amounts of metabolites in human hepatocytes. As summarized in Tables 6A and 6B below, the proteo-(non-deuterium enriched) form of NS2 was significantly metabolized over the course of only 120 min by rat, dog, monkey, or human hepatocytes. For example, dog hepatocytes metabolized NS2 into two different mono-oxidation products (M1 and M2) and two different glutathione (GSH) conjugates (M5 and M6), as evaluated by LC retention times and mass spectrometry data (see FIG. 3 ). Cyno monkey hepatocytes metabolized NS2 into a mono-oxidation metabolite (M1), four oxidation+glucuronidation metabolites (M3, M4, M7, and M9), and a glucuronidation metabolite (M8) (see FIG. 2 ). Human hepatocytes metabolized NS2 into a mono-oxidation metabolite (M1), two oxidation+glucuronidation metabolites (M7 and M9), and a glucuronidation metabolite (M8) (see FIG. 1 ).

TABLE 6A Metabolite Profiling of NS2 in Hepatocytes: Data Summary Retention Observed Metabolites (T-120 min) Time m/z Possible Cyno Analyte (min) m/z shift Biotransformation Rat Dog Monkey Human NS2 4.4 237 — Parent NA NA NA NA M1 2.1 253  +16 mono-oxidation ++++ ++++ ++++ ++++ M2 2.9 253  +16 mono-oxidation + M3 3.0 429 +192 oxidation + + glucuronidation M4 3.2 429 +192 oxidation + ++ glucuronidation M5 3.5 542 +305 GSH-conjugation + M6 3.7 542 +305 GSH-conjugation ++ M7 1.7 429 +192 oxidation + trace ++ + glucuronidation M8 3.98 413 +176 glucuronidation trace ++ ++ M9 3.9 429 +192 oxidation + + + glucuronidation m/z: Mass-to-Charge ratio of analyte NA = Not applicable Relative degree of observed metabolite formation is denoted by “+”, with ++++ being the most abundant metabolite (assuming that the ionization potential of the parent is similar to that of the metabolites)

TABLE 6B Peak Areas of Observed Metabolites Peak Areas Observed Metabolites (T-120 mm) Cyno Analyte Rat Dog Monkey Human NS2 3.14E+08 4.79E+07 1.74E+08 4.41E+08 M1 4.25E+08 2.83E+08 4.47E+08 2.57E+08 M2 1.58E+07 M3 7.50E+06 M4 1.92E+07 M5 1.69E+07 M6 7.67E+07 M7 trace 1.35E+07 1.62E+07 M8 trace 3.15E+07 4.67E+07 M9 1.22E+07 1.51E+07

Mass spectral analysis of NS2, each metabolite M1-M9, deuterated NS2, and the metabolite produced in human hepatocytes from deuterated NS2 are shown in FIGS. 6-17 .

In contrast to non-deuterium-enriched NS2, exposure of deuterium-enriched NS2 (i.e., compound I-1, or NS2-D6 in Table 7) to human hepatocytes for 120 min resulted in mono-oxidation metabolite M1 as the sole detectable metabolite (see Table 7 and FIGS. 5 and 17 ). The amount of M1 produced was also greatly reduced relative to that produced when non-deuterated NS2 was exposed to human hepatocytes (compare FIGS. 1 and 5 ). Such a dramatic reduction in metabolite production could not have been predicted in advance.

TABLE 7 Metabolite Profiling of Deuterated NS2 in Human Hepatocytes: Data Summary Retention Observed Metabolites Time m/z Possible (T-120 min) Analyte (min) m/z shift Biotransformation Human NS2-D6 4.4 243 — Parent NA M1 2.1 259 +16 mono-oxidation ++ m/z: Mass-to-Charge ratio of analyte NA = Not applicable Relative degree of observed metabolite formation is denoted by “+”, with ++++ being the most abundant metabolite (assuming that the ionization potential of the parent is similar with the metabolites) Note: Based on the number of observed metabolites, deuterated-NS2 exhibited less metabolism over the course of 2 hours relative to the non-deuterated NS2 molecule.

The results shown in Table 7 are also surprising in that the reduction in the number and amount of metabolites may not simply be due to deuterium incorporation making an enzymatically-catalyzed rate-determining step more difficult. Rather, the observed m/z of 259 is consistent with all six deuterium atoms being retained in the mono-oxidation product. Without wishing to be bound by theory, this may indicate that the deuterium enrichment is influencing metabolism at a remote location on the molecule, and perhaps not simply through, e.g., a primary kinetic isotype effect.

References: 1) McGinnity, D. F. et al. (2004). “Evaluation of fresh and cryopreserved hepatocytes as in vitro drug metabolism tools for the prediction of metabolic clearance.” Drug Metab. Dispos. 32(11):1247-1253. 2) Sahi, J. et al. (2010). “Hepatocytes as a tool in drug metabolism, transport and safety evaluations in drug discovery.” Current Drug Discov. Technol. 7(3): 188-198.

Example 12: Evaluation of Dose Responses for Protective Activity from Hydrogen Peroxide Toxicity in Dissociated Hippocampal Cultures for NS2 (i.e. Non-Deuterated NS2)

Experimental Plan for NS2 Dose Response Evaluation for Protection from Hydrogen Peroxide Toxicity:

A. Test Agent: NS2 FW: 236

1. Source 1: CoreRx lot 093-FOR CNS2; amount used: 6.4 mg; micro-milled sample (average particle size is about 16 micron); was derived from J-Star Lot BR-NS2-11-01

2. Source 2: J-Star lot BR-NS2-1; amount used: 6 mg; non-milled sample

B. Formulations and Stock Solution Preparation

Two types of formulation were compared: dimethyl sulfoxide (DMSO) and Captisol®

1. Captisol® formulation: Captisol® was dissolved at 5 mg/ml (i.e., at 0.5%) in Dulbecco's Phosphate Buffered Saline (DPBS). A 10 mM NS2 stock solution (stock A) was prepared by dissolving 1 mg (4.24 mol) into 0.42 ml of the Captisol® solution to give an initial stock of 10 μmol/ml or 10 mM (i.e., 2.38 mg/mL). The stock solution was clear after vortex mixing.

2. DMSO formulation: DMSO was used as a comparator to Captisol®. Five mg (21.12 mol) of NS2 was dissolved in 0.21 mL of DMSO for a 100 mM stock solution (i.e., 23.8 mg/mL). The 100 mM NS2 DMSO formulation was clear. Log dilutions were done with DPBS. Upon dilution 1:10 to 10 mM with DPBS, the solution became cloudy, but, cleared after extensive vortex mixing. In general, our benchmark concentration goal for DMSO in primary neuronal cultures is less than 0.1%, to avoid pharmacological actions of DMSO. Note that 1% DMSO was used for the 1 mM test concentration of NS2. No apparent toxicity was observed in the assays in the 5 hr test (see results from Experiment 9 below).

C. Details on the Preparation of Stock Solutions

NS2 in Captisol®:

1. Stock A was 10 mM NS2 in 0.5% Captisol®. Added 10 μl into 100 μl for a final concentration of 1 mM NS2, in 0.05% Captisol®, in each well.

2. Stock B was prepared by adding 50 μl of stock A to 450 μl of DPBS to yield a 1 mM solution of NS2. Added 10 μl into 100 μl DPBS for a final concentration of 100 μM NS2 in 0.005% Captisol®.

3. Stock C was prepared by adding 50 μl of stock B into 450 μl of DPBS to yield a 100 μM solution of NS2. Added 10 μl into 100 μl DPBS for a final concentration of 10 μM NS2 in 0.0005% Captisol®.

4. Stock D was prepared by adding 50 μl of stock C into 450 μl of DPBS to yield a 10 μM solution of NS2. Added 10 μl into 100 μl DPBS for a final concentration of 1 μl NS2 in 0.00005% Captisol®

5. Stock E was prepared by adding 50 μl of stock D into 450 μl of DPBS to yield a 1 μM solution of NS2. Added 10 μl into 100 μl DPBS for a final concentration of 0.1 μM NS2 in 0.000005% Captisol®.

NS2 in DMSO:

1. Stock A was 100 mM NS2 in 100% DMSO.

2. Stock B was prepared by adding 50 μl of stock A into 450 μl of DPBS for a final concentration of 10 mM NS2. Added 10 μl into 100 μl DPBS to yield a final concentration of 1 mM NS2 in 1% DMSO.

3. Stock C was prepared by adding 50 μl of stock B into 450 μl of DPBS for a final concentration of 1 mM NS2. Added 10 μl into 100 μl DPB S to yield a final concentration of 100 NS2 in 0.1% DMSO.

4. Stock D was prepared by adding 50 μl of stock C into 450 μl of DPBS for a final concentration of 100 μM NS2. Added 10 μl into 100 μl DPB S to yield a final concentration of 10 NS2 in 0.01% DMSO.

5. Stock E was prepared by adding 50 μl of stock D into 450 μl of DPBS for a final concentration of 10 μM NS2. Added 10 μl into 100 μl DPBS to yield a final concentration of 1 NS2 in 0.001% DMSO.

6. Stock F was prepared by adding 50 μl of stock E into 450 μl of DPBS for a final concentration of 1 μM NS2. Added 10 μl into 100 μl DPBS for a final concentration of 0.1 μM NS2 in 0.0001% DMSO.

In all cases, 10 μl of the appropriate dilution was added to 100 μl for a total volume of 110 μl in the well.

D. Culture Conditions Designed to Detect NS2-Mediated Neuroprotection from Oxidative Stress Associated with Hydrogen Peroxide

1. Rat hippocampal cultures were prepared as previously described (Brenneman D E, Smith G R, Zhang Y, Du Y, Kondaveeti S K, Zdilla M J, Reitz A B. (2012) J. Molecular Neuroscience, 47:368-379). Under these conditions, the cultures are at least 90% neuronal. The most abundant non-neuronal cells are astrocytes.

2. All cultures were prepared into a 96-well format at a plating density of 10K cells per well. Cultures were treated between day 10 and day 21 after dissociation of E18 hippocampal tissue. For these experiments, all plates were treated on day 13. In all experiments, the hydrogen peroxide was added to the cultures 10 minutes after treatment with NS2 or cannabidiol (CBD). For each treatment condition, the number of replicates was five.

3. All cultures were plated in B27/Neural Basal Medium. On the day of treatment, all cultures were given a complete change of medium into B27/Neural Basal Medium without antioxidants.

4. As previously determined (Brenneman et al., 2012), 10 μM hydrogen peroxide was used to produce toxicity and oxidative stress. As described previously [Jarrett, S G, Liang, L-P, Hellier, J L, Staley, K J and Patel, M. (2008) Neurobiol. Dis 30(1): 130-138], 10 μM hydrogen peroxide has been observed in the hippocampus of rats with a kainate model of status epilepticus.

5. The positive control used in all studies was 10 μM cannabidiol (CBD), a known antioxidant agent [Hampson et al. (1998), Proc. Nat. Acad. Sci 95:8268-8273] that is protective against oxidative stress in primary neurons [Brenneman, D E, Petkanas, D and Kinney, W. A. (2014) Annual Symposium on the Cannabinoids, page 129].

6. Neither the negative control wells, the hydrogen peroxide wells, nor the positive control wells contained any drug vehicle.

E. Assays

Both assays used in this study have been described in detail [Brenneman D E, Smith G R, Zhang Y, Du Y, Kondaveeti S K, Zdilla M J, Reitz A B. (2012) J. Molecular Neuroscience, 47:368-379].

1. The CFDA neuronal viability assay. In this assay, the CFDA dye is taken up by all live cells and cleaved by esterases to release fluorescein. The neuronal specificity is achieved because neurons cannot remove this dye, whereas efflux of the dye from non-neuronal cells can occur over time. After washing away the extracellular dye, the cultures were read in a fluorimeter; intracellular dye intensity is proportional to the live neuronal population. Original reference: Petroski, R E and Geller H M. (1994) Selective labeling of embryonic neurons cultures on astrocyte monolayers with 5(6)-carboxyfluorescein diacetate (CFDA) J. Neurosci. Methods 52:23.32. The mean control level for each experiment is shown as a longdashed reference line.

2. A cell death assay, using propidium iodide, was conducted simultaneously with the CFDA assay in the same well. This dye is excluded from live cells and binds to the DNA of dead cells. The assay detects both necrotic and apoptotic cell death; it does not distinguish between neuronal cell death and non-neuronal cell death. See Sarafian T A, Kouyoumjian S, Tashkin D, Roth M D. (2002) Tox. Letters. 133: 171-179. The mean control level is shown as a medium-dashed reference line.

3. Reagents used:

-   -   a. Hydrogen Peroxide solution, 30 wt %; Sigma-Aldrich         (216736-100 ml, Lot MKBV382V)     -   b. Captisol® (Lot 17CX01-HQ-00088) provided by Aldeyra         Therapeutics     -   c. Dimethyl Sulfoxide; Sigma-Aldrich (472301-100 ml) Batch 21096         JK     -   d. Propidium Iodide Sigma-Aldrich (P4864-10 ml; 1 mg/ml solution         in water)     -   e. CFDA [5(6)-Carboxyfluorescein Diacetate] Sigma-Aldrich         Product Number: 21879-100 mg-F     -   f. Cannabidiol solution, 10 mg/ml in ethanol; Sigma-Aldrich         Product Number: 90899-1 ml     -   g. Dulbecco's Phosphate Buffered Saline. Gibco (14190-144) Lot         1165767

4. Data Analyses

-   -   a. Data Acquisition: Data were stored on Advanced Neural         Dynamics computers for analyses. Data acquisition was performed         on Cytofluor Fluorimeter and transferred to Excel spreadsheet         for analysis with Sigma Plot 11.     -   b. Statistical Analysis: All data were statistically analyzed by         an Analysis of Variance with the Multiple Comparisons versus         Control Group (Holm-Sidak) method. Statistical significance was         taken at the P<0.05 level. In all cases, comparisons were made         to the negative control (10 μM hydrogen peroxide treatment).     -   c. Methodology for EC₅₀ determination:         -   i. A broad concentration range was chosen to screen NS2 in             an EC₅₀ potency analysis. A log-based concentration series             from 0.1 μM to 1 mM was used, recognizing that further             analysis involving half-log concentration may be necessary             to assess EC₅₀s.         -   ii. A nonlinear regression analysis was used to determine             the equation of the line that best fits the data. (Four             parameter Logistic curve)         -   iii. Based on the Logistic equation below, the EC₅₀s for             neuroprotection were calculated and plotted by SigmaPlot 11             to determine the concentration required to produce             half-maximal responses for both assays. Drop lines were used             to show the axes intersections determining the EC₅₀.

${{Four}{Parameter}{Logistic}{Equation}}{y = {\min + \frac{\max - \min}{1 + \left( \frac{x}{{EC}50} \right)^{- {Hillslope}}}}}$

-   -   -   -   This results in a typical dose-response curve with a                 variable slope parameter. It is sometimes abbreviated as                 4PL. The four parameters are: min (bottom of the curve),                 max (top of the curve). EC₅₀=Concentration of ligand                 that produced 50% of maximal effective response.

5. Summary of Results

Table 8 shows a summary of protection studies for NS2 (i.e., non-deuterated NS2) in rat hippocampal cultures.

TABLE 8 Summary of Protection Studies for N52 in Rat Hippocampal Cultures Full No Efficacy effect Formu- Concen- Concen- Assay Compound lation tration** tration EC50 ± SE CFDA NS2 CoreRx Captisol ® 100 μM 1 μM 6.8 ± 3.6 μM CFDA NS2 CoreRx DMSO 100 μM 1 μM 9.8 μM * CFDA NS2 J-Star Captisol ® 100 μM 1 μM 9.1 ± 2.8 μM CFDA NS2 J-Star DMSO 100 μM 1 μM 2.6 μM * PI NS2 CoreRx Captisol ®  10 μM 1 μM 1.3 μM * PI NS2 CoreRx DMSO  10 μM 1 μM 1.3 μM * PI NS2 J-Star Captisol ®  10 μM 1 μM 2.8 μM * PI NS2 J-Star DMSO  10 μM 1 μM 1.1 μM * CFDA None Captisol ® Not active PI None DMSO Not active * Because of the steep nature of the logistic curve observed for these data sets, further analysis using half-log concentrations may be necessary to determine the EC₅₀ under these conditions. The posted values should be considered estimates. **Concentration of test agent showing assay response levels not significantly different from that of no treatment controls.

6. Graphical Analyses of Experimental Findings and Raw Data

a. Experiment 1: Dose response to micro-milled NS2 (CoreRx) in Captisol®. Effect on neuronal viability after co-treatment with 10 μM hydrogen peroxide.

-   -   i. NS2 Source: CoreRx, micro-milled     -   ii. Formulation: Initial stock was formulated in 0.5% Captisol®     -   iii. Assay: CFDA     -   iv. Toxin: 10 μM hydrogen peroxide     -   v. Duration of treatment: 5 hours     -   vi. Growth medium: B27/neurobasal medium without antioxidants     -   vii. Culture matrix: poly-L-lysine     -   viii. Conclusions: The EC₅₀ was observed at 6.8 μM; full         efficacy relative to controls (CBD+HP and no-treatment control)         was observed at 100 μM NS2.

NS2 is fully neuroprotective against hydrogen peroxide toxicity at 100 μM in this assay. Results are shown in Table 9 and FIGS. 18 and 19 .

TABLE 9 Effect on Neuronal Viability After Co-Treatment with 10 μM Hydrogen Peroxide Statistical CBD + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP HP Analysis Control 10 μM 1 mM 100 μM 10 μM 1 μM 0.1 μM 10 μM 45216 45216 45216 55866 41262 31538 31107 24829 42919 53439 45216 47620 27827 24829 29842 27827 47620 50134 35167 45216 48549 30050 27827 29430 47620 52763 53439 45216 39139 23428 19585 25008 48706 42919 35405 38622 31107 38622 26294 28320 Mean  46416*  48894*  42889*  46508*  37577* 29693 26931 27083 Std Error  1044  2078  3448  2778  3697  2703  2014  921 P value* <0.001 <0.001 <0.001 <0.001 <0.002 N.S. N.S. N.S. % of Control 100 ± 2 105 ± 4 92 ± 07 100 ± 6 80 ± 8 64 ± 6 58 ± 4 58 ± 2 * Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

b. Experiment 2: Dose response to micro-milled NS2 (CoreRx) in Captisol®. Effect on cell death after co-treatment with 10 μM hydrogen peroxide.

-   -   i. NS2 Source: CoreRx (micro-milled)     -   ii. Formulation: Initial stock was formulated in 0.5% Captisol®     -   iii. Assay: Propidium Iodide     -   iv. Toxin: 10 μM hydrogen peroxide     -   v. Duration of treatment: 5 hours     -   vi. Growth medium: B27/neurobasal medium without antioxidants     -   vii. Culture matrix: poly-L-lysine     -   viii. Conclusions: The EC₅₀ was observed at 1.3 μM; full         efficacy relative to controls (CBD+HP and no-treatment control)         was observed at 10 μM NS2.

NS2 is fully neuroprotective against hydrogen peroxide toxicity with this assay. These data suggest that the protective effect against cell death may be slightly more potent than that observed for neuronal viability. Results are shown in Table 10 and FIGS. 20 and 21 .

TABLE 10 Effect of NS2 (CoreRx) on Cell Death in Hippocampal Cultures After Co- treatment with 10 μM Hydrogen Peroxide for 5 hours Statistical CBD + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP HP Analysis Control 10 μM 1 mM 100 μM 10 μM 1 μM 0.1 μM 10 μM 140 144 246 144 180 319 246 315 180 121 184 180 57 246 246 306 137 192 180 192 246 254 246 209 144 117 188 246 180 246 246 346 180 180 217 137 180 267 323 267 Mean 156*   151*   203*   180*   169*   266 261 289 Std Error 10 15 13 20 31 14 15 24 P value* <0.001 <0.001 <0.003 <0.001 <0.001 N.S. N.S. N.S. % of Control 100 ± 6 97 ± 9 130 ± 8 115 ± 13 108 ± 20 171 ± 9 167 ± 10 185 ± 15 * Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

c. Experiment 3: Dose response to micro-milled NS2 (CoreRx) in DMSO. Effect on neuronal viability after co-treatment with 10 μM hydrogen peroxide.

-   -   i. NS2 Source: CoreRx (micro-milled)     -   ii. Formulation: Initial stock was formulated in 100% DMSO     -   iii. Assay: CFDA     -   iv. Toxin: 10 μM hydrogen peroxide     -   v. Duration of treatment: 5 hours     -   vi. Growth medium: B27/neurobasal medium without antioxidants     -   vii. Culture matrix: poly-L-lysine     -   viii. Conclusions: The EC₅₀ was observed at 9.8 μM; full         efficacy relative to controls (CBD+HP and no-treatment control)         was observed at 100 μM NS2.

The use of DMSO as a formulation produced an EC₅₀ that was very similar to that observed with Captisol®. NS2 is fully neuroprotective against hydrogen peroxide toxicity in this assay. Results are shown in Table 11 and FIGS. 22 and 23 .

TABLE 11 Effect of NS2 (CoreRx; in DMSO) on Neuronal Viability After Co- Treatment with 10 mM Hydrogen Peroxide for 5 hours Statistical CBD + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP HP Analysis Control 10 μM 1 mM 100 μM 10 μM 1 μM 0.1 μM 10 μM 47420 49934 38422 42719 38422 20765 23428 20844 38939 48038 36415 40792 32661 32886 26094 31447 40792 42719 42719 42439 32661 32886 27627 31338 39199 42719 39139 53580 32661 23228 25189 24899 48706 38422 36415 37108 34496 23400 29230 23228 Mean 43011*   44366*   38622*   43328*   34180*   26633 26314 26351 Std Error 2097 2064 1158 2751 1118 2595 997 2157 P value* <0.001 <0.001 <0.001 <0.001 <0.009 N.S. N.S. N.S. % of Control 100 ± 5 103 ± 5 90 ± 3 101 ± 6 79 ± 3 62 ± 6 61 ± 2 61 + 5 * Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

d. Experiment 4: Dose response to micro-milled NS2 (CoreRx) in DMSO. Effect on cell death after co-treatment with 10 μM hydrogen peroxide.

-   -   i. NS2 Source: CoreRx (micro-milled)     -   ii. Formulation: Initial stock was formulated in 100% DMSO     -   iii. Assay: Propidium Iodide     -   iv. Toxin: 10 μM hydrogen peroxide     -   v. Duration of treatment: 5 hours     -   vi. Growth medium: B27/neurobasal medium without antioxidants     -   vii. Culture matrix: poly-L-lysine     -   viii. Conclusions: The EC₅₀ was observed at 1.3 μM; full         efficacy relative to controls (CBD+HP and no-treatment control)         was observed at 10 μM NS2.

NS2 is fully neuroprotective against hydrogen peroxide toxicity with this assay. These data suggest that the protective effect against cell death may be slightly more potent than that observed for neuronal viability. Results are shown in Table 12 and FIGS. 24 and 25 .

TABLE 12 Effect of NS2 (CoreRx in DMSO) on Cell Death in Hippocampal Cultures After Co-Treatment with 10 μM Hydrogen Peroxide Statistical CBD + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP HP Analysis Control 10 μM 1 mM 100 μM 10 μM 1 μM 0.1 μM 10 μM 117 180 204 125 180 254 246 315 133 180 180 180 188 263 263 246 144 117 221 180 192 254 254 346 180 125 188 192 117 254 315 246 129 117 180 117 137 315 259 259 Mean 141*   144*   195*   159*   163*   268 267 282 Std Error 11 15 8 16 15 12 12 20 P value* <0.001 <0.001 <0.001 <0.001 <0.001 N.S. N.S. N.S. % of Control 100 ± 8 102 ± 11 138 ± 6 113 ± 11 115 ± 11 190 ± 9 189 ± 9 200 ± 14 * Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

e. Experiment 5: Dose response to non-milled NS2 (J-Star) in Captisol®. Effect on neuronal viability after co-treatment with 10 μM hydrogen peroxide.

-   -   i. NS2 Source: J-Star (non-milled)     -   ii. Formulation: Initial stock was formulated in 0.5% Captisol®     -   iii. Assay: CFDA     -   iv. Toxin: 10 μM hydrogen peroxide     -   v. Duration of treatment: 5 hours     -   vi. Growth medium: B27/neurobasal medium without antioxidants     -   vii. Culture matrix: poly-L-lysine     -   viii. Conclusions: The EC₅₀ was observed at 9.1 μM; full         efficacy relative to controls (CBD+HP and no-treatment control)         was observed at 100 μM NS2.

NS2 is fully neuroprotective against hydrogen peroxide toxicity with this assay. Results are shown in Table 13 and FIGS. 26 and 27 .

TABLE 13 Dose Response Data for Non-Milled NS2 (J-Star) in Captisol ® Showing Effect on Neuronal Viability after Co-Treatment with 10 μM Hydrogen Peroxide Statistical CBD + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP HP Analysis Control 10 μM 1 mM 100 μM 10 μM 1 μM 0.1 μM 10 μM 40722 36415 38422 36415 37407 22135 21242 25171 41535 38422 40722 31538 36861 24829 26094 21889 41198 41884 38622 29230 32861 26094 22053 23428 40992 43711 43200 42919 26756 28021 22089 19885 36415 38809 36615 47620 25171 20609 29230 21162 Mean 40172*   40858*   39516*   37544*   31811*   24338 24142 22307 Std Error 949 5136 1128 3442 2525 1335 1528 917 P value* <0.001 <0.001 <0.001 <0.001 <0.001 N.S. N.S. N.S. % of Control 100 ± 2 102 ± 12 98 ± 3 93 ± 9 79± 6 61 ± 3 60 ± 4 56 ± 2 * Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

f. Experiment 6: Dose response to non-milled NS2 (J-Star) in Captisol®. Effect on cell death after co-treatment with 10 μM hydrogen peroxide.

-   -   i. NS2 Source: J-Star (non-milled)     -   ii. Formulation: Initial stock was formulated in 0.5% Captisol®     -   iii. Assay: Propidium Iodide     -   iv. Toxin: 10 μM hydrogen peroxide     -   v. Duration of treatment: 5 hours     -   vi. Growth medium: B27/neurobasal medium without antioxidants     -   vii. Culture matrix: poly-L-lysine     -   viii. Conclusions: The EC₅₀ was observed at 2.8 μM; full         efficacy relative to controls (CBD+HP and no-treatment control)         was observed at 10 μM NS2.

NS2 is fully neuroprotective against hydrogen peroxide toxicity with this assay. These data suggest that the protective effect against cell death may be slightly more potent than that observed for neuronal viability. Results are shown in Table 14 and FIGS. 28 and 29 .

TABLE 14 Dose Response Data for Non-Milled NS2 (J-Star) in Captisol ® Showing Effect on Cell Death after Co-Treatment with 10 μM Hydrogen Peroxide Statistical CBD + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP HP Analysis Control 10 μM 1 mM 100 μM 10 μM 1 μM 0.1 μM 10 μM 140 180 180 129 117 184 204 271 144 144 133 129 196 315 180 323 140 117 172 125 117 337 246 246 117 121 180 180 180 180 350 246 164 200 180 133 188 192 180 263 Mean 141*   152*   169*   139*   160*   242 232 270 Std Error 7 16 9 10 18 35 32 14 P value* <0.001 <0.001 <0.001 <0.001 <0.001 N.S. N.S. N.S. % of Control 100 ± 5 108 ± 11 120 ± 6 99 ± 7 113 ± 12 172 ± 24 165 ± 22 191 ± 10 *Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

g. Experiment 7: Dose response to non-milled NS2 (J-Star) in DMSO. Effect on neuronal viability after co-treatment with 10 μM hydrogen peroxide.

-   -   i. NS2 Source: J-Star (non-milled)     -   ii. Formulation: Initial stock was formulated in 100% DMSO     -   iii. Assay: CFDA     -   iv. Toxin: 10 μM hydrogen peroxide     -   v. Duration of treatment: 5 hours     -   vi. Growth medium: B27/neurobasal medium without antioxidants     -   vii. Culture matrix: poly-L-lysine     -   viii. Conclusions: The EC₅₀ was observed at 2.6 μM; full         efficacy relative to controls (CBD+HP and no-treatment control)         was observed at 100 μM NS2.

NS2 is fully neuroprotective against hydrogen peroxide toxicity with this assay. Results are shown in Table 15 and FIGS. 30 and 31 .

TABLE 15 Dose Response Data for Non-Milled NS2 (J-Star) in DMSO Showing Effect on Neuronal Viability after Co-Treatment with 10 μM Hydrogen Peroxide Statistical CBD + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP HP Analysis Control 10 μM 1 mM 100 μM 10 μM 1 μM 0.1 μM 10 μM 40722 47620 45216 42919 31214 20809 23600 19585 42919 34696 39269 38622 33768 29430 25371 24829 42919 48238 26576 38622 33425 23257 31107 25462 43342 41535 26294 26294 32194 34696 22089 22089 38622 36615 45807 52763 46105 29430 21926 23772 Mean 41705*   41741*   36632*   39844*   35341*   27524 24819 23147 Std Error 897 2763 4318 4260 2729 2470 1690 1058 P value * <0.001 <0.001 <0.002 <0.001 <0.004 N.S. N.S. N.S. % of Control 100 ± 2 100 ± 7 88 ± 10 96 ± 10 85 ± 7 66 ± 6 60 ± 4 56 + 3 *Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

h. Experiment 8: Dose response to non-milled NS2 (J-Star) in DMSO. Effect on cell death after co-treatment with 10 μM hydrogen peroxide.

-   -   i. NS2 Source: J-Star (non-milled)     -   ii. Formulation: Initial stock was formulated in 100% DMSO     -   iii. Assay: Propidium Iodide     -   iv. Toxin: 10 μM hydrogen peroxide     -   v. Duration of treatment: 5 hours     -   vi. Growth medium: B27/neurobasal medium without antioxidants     -   vii. Culture matrix: poly-L-lysine     -   vii. Conclusions: The EC₅₀ was observed at 1.1 μM; full efficacy         relative to controls (CBD+HP and no-treatment control) was         observed at 10 μM NS2.

NS2 is fully neuroprotective against hydrogen peroxide toxicity with this assay. These data suggest that the protective effect against cell death may be slightly more potent than that observed for neuronal viability. Results are shown in Table 16 and FIGS. 32 and 33 .

TABLE 16 Dose Response Data for Non-Milled NS2 (J-Star) in DMSO Showing Effect on Cell Death after Co-Treatment with 10 μM Hydrogen Peroxide Statistical CBD + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP NS2 + HP HP Analysis Control 10 μM 1 mM 100 μM 10 μM 1 μM 0.1 μM 10 μM 180 180 180 125 180 246 246 254 117 133 204 125 180 196 315 246 180 117 133 246 172 246 204 319 164 129 117 246 180 188 350 246 144 184 250 133 192 250 259 259 Mean 157*   161*   177*   175*   181*   225 275 265 Std Error 12 12 24 29 3 14 26 14 P value * <0.001 <0.001 <0.002 <0.002 <0.003 N.S. N.S. N.S. % of Control 100 ± 8 103 ± 8 113 ± 15 111 ± 18 115 ± 2 143 ± 9 175 ± 17 169 ± 9 *Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

i. Experiment 9: Dose response to Formulation vehicles. Effect on neuronal viability after co-treatment with 10 μM hydrogen peroxide.

-   -   i. Test agents: Captisol® [Cap; (5 mg/ml i.e., 0.5%)] and 1%         DMSO (abbreviated to DM; 1% was the highest concentration used).         The amounts used for the formulations were matched to those used         in the NS2 studies.     -   ii. Assay: CFDA     -   iii. Toxin: 10 μM hydrogen peroxide     -   iv. Duration of treatment: 5 hours     -   v. Growth medium: B27/neurobasal medium without antioxidants     -   vi. Culture matrix: poly-L-lysine     -   vii. Conclusions: There was no detectible effect on neuronal         viability from either Captisol® or DMSO when tested under the         same conditions as for NS2. Results are shown in Tables 17 and         18 and FIG. 34 .

TABLE 17 Dose Response Data for Formulation Vehicles Showing Effect on Neuronal Viability after Co-Treatment with 10 μM Hydrogen Peroxide Statistical CBD + HP Cap + HP Cap + HP Cap + HP Cap + HP Cap + HP HP Analysis Control 10 μM 500 μγ/ml 50 μg/ml 5 μg/ml 500 pg/ml 50 pg/ml 10 μM 52563 52563 24629 26471 23833 23228 27627 24629 34496 34496 29230 24629 25171 28219 23833 26094 49934 42719 20687 27627 23833 25171 24718 23228 38422 42719 31107 26188 27627 26471 26094 21889 45016 45216 29746 23228 29230 23572 26094 27627 Mean 44086*   43543*   27080 25629 25939 25332 25673 24693 Std Error 3398 2893 1934 767 1076 927 651 1014 P value * <0.001 <0.001 N.S. N.S. N.S. N.S. N.S. N.S. % of Control 100 ± 8 99 ± 7 61 ± 4 58 ± 2 59 ± 2 57 ± 2 58 ± 2 56 ± 2 *Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

TABLE 18 Dose Response Data for Formulation Vehicles Showing Effect on Neuronal Viability after Co-Treatment with 10 μM Hydrogen Peroxide Statistical CBD + HP DM + HP DM + HP DM + HP DM + HP DM + HP HP Analysis Control 10 μM 1% 0.1% .01% .001% .0001% 10 μM 52563 52563 24629 31665 24629 21003 23228 22218 34496 34496 24629 25171 26094 32661 28219 23428 49934 42719 24629 32886 24629 29230 19685 24899 38422 42719 27627 22089 26094 18215 19385 21889 45016 45216 27627 29333 24629 18215 21971 26482 Mean 44086*   43543*   25828 28229 25215 23865 22498 23783 Std Error 3398 2893 734 2022 359 2985 1599 857 P value * <0.001 <0.001 N.S. N.S. N.S. N.S. N.S. N.S. % of Control 100 ± 8 99 ± 7 59 ± 2 64 ± 5 57 ± 1 54 ± 7 51 ± 4 54 ± 2 *Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

j. Experiment 10: Dose response to Formulation vehicles. Effect on cell death after co-treatment with 10 μM hydrogen peroxide.

-   -   i. Test agents: Captisol® [CP (5 mg/ml i.e., 0.5%)] and DMSO         (abbreviated to DM; 1% was the highest used)     -   ii. Assay: Propidium Iodide     -   iii. Toxin: 10 μM hydrogen peroxide     -   iv. Duration of treatment: 5 hours     -   v. Growth medium: B27/neurobasal medium without antioxidants     -   vi. Culture matrix: poly-L-lysine     -   vii. Conclusions: There was no detectible effect on cell death         from either Captisol® or DMSO when tested under the same         conditions as for NS2. Results are shown in Tables 19 and 20 and         FIG. 35 .

TABLE 19 Dose Response Data for Formulation Vehicles Showing Effect on Cell Death after Co-Treatment with 10 μM Hydrogen Peroxide Statistical CBD + HP Cap + HP Cap + HP Cap + HP Cap + HP Cap + HP HP Analysis Control 10 μM 500 μg/ml 50 μg/ml 5 mg/ml 500 pg/ml 50 pg/ml 10 μM 125 140 246 246 246 246 229 263 144 133 246 254 250 246 229 188 117 180 263 180 140 180 246 246 180 184 250 350 246 315 246 246 144 156 250 315 288 319 246 250 Mean 142*   159*   251 269 234 261 239 239 Std Error 11 10 3 29 25 26 4 13 P value * <0.001 <0.003 N.S. N.S. N.S. N.S. N.S. N.S. % of Control 100 ± 8 112 ± 7 177 ± 2 189 ± 20 165 ± 18 184 ± 18 168 ± 3 168 ± 9 Cap = Captisol ® vehicle. *Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

TABLE 20 Dose Response Data for Formulation Vehicles Showing Effect on Cell Death after Co-Treatment with 10 μM Hydrogen Peroxide Statistical CBD + HP DM + HP DM + HP DM + HP DM + HP DM + HP HP Analysis Control 10 μM 1% 0.1% .01% .001% .0001% 10 μM 125 140 250 297 276 267 246 267 144 133 246 297 267 259 246 254 117 180 315 276 246 246 184 246 180 184 230 192 250 246 267 246 144 156 267 276 246 246 225 246 Mean 142*   159*   262 268 257 253 234 252 Std Error 11 10 15 19 6 4 14 4 P value * <0.001 <0.003 N.S. N.S. N.S. N.S. N.S. N.S. % of Control 100 ± 8 112 ± 7 185 ± 11 189 ± 13 181 ± 4 178 ± 3 165 ± 10 177 ± 3 DM = DMSO vehicle. *Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

7. Summary of Observations and Conclusions

A. NS2 exhibited neuroprotective activity against hydrogen peroxide toxicity in the CFDA assay, with both formulations (DMSO and Captisol®) and with both compound batches (CoreRx and J-Star, milled and non-milled, respectively).

B. The neuroprotective effect of NS2 in the CFDA assay was equal to the control (no HP treatment) and positive control (CBD), indicating full protection.

C. Full protection relative to controls was observed with both NS2 formulations and NS2 batches at 100 μM NS2 in the CFDA neuronal viability assay, whereas the no effect concentration for both formulations and batches was 1 μM.

D. Non-linear curve fitting Logistic analyses indicated that NS2 EC₅₀s in the CFDA neuronal viability assay ranged from 3 to 10 μM. The best available EC₅₀ estimate is from the CoreRx NS2 (milled) formulated in Captisol®, which showed an EC₅₀ of 7±4 μM. All the EC₅₀s from the different NS2 formulations for the CFDA assays fall within this range. It is our conclusion that the two compound batches and the two formulations are characterized by their substantial similarity.

E. NS2 exhibited protective activity against hydrogen peroxide toxicity in the Propidium Iodide (PI) assay. This was observed with both formulations and with both compound batches.

F. The protective effect of NS2 in the PI assay was equal to that of the control (no HP treatment) and positive control (CBD), indicating full protection.

G. Full protection relative to no-treatment controls was observed with both NS2 formulations and NS2 batches at 10 μM NS2 in the PI assay, whereas the no effect concentration for both formulations and batches was 1 μM. It was a consistent finding that the NS2 response in the cell death assay exhibited greater potency for full protection than in the CFDA assay. It should be recognized that the cell death assay is not specific to neurons and may involve non-neuronal cells that are present in this model CNS system.

H. Non-linear Logistic curve fitting indicated that EC₅₀s for the PI assay ranged from 1.1 to 2.8 μM. However, the steep nature of the Logistic curve with this assay made estimates difficult without the measurement of half-log concentration responses to help define the inflection point of the curves. The best available estimate is the mean value for all PI data, which is 2±1 μM. It is our conclusion that the two compound batches and the two formulations were characterized by their substantial similarity. Because of the narrow response ranges in the PI assay, further analysis may be required to refine the EC₅₀ estimate. These data suggest that NS2 may be more potent in preventing cell death than in increasing neuronal viability against hydrogen peroxide toxicity.

I. The toxic signal produced by 10 μM hydrogen peroxide was typical of a wide variety of oxidative stressors (ethanol, heavy metals, ammonium acetate, and glutamate) that have been tested in the past, with decreases from control ranging from 30 to 50%.

J. The positive control (10 μM cannabidiol) was active on every test plate, indicating that the model system was responding in a typical manner.

Example 13: Dose Responses for Three Deuterated Compounds Assessing Protective Activity from Hydrogen Peroxide Toxicity in Dissociated Hippocampal Cultures

A. Experimental Plan for Dose Response Evaluation for Protection from Hydrogen Peroxide Toxicity.

1. Test Agents:

-   -   a. ALD-6-batch 1 (Legacy ID: NS2-D6 or compound I-1); amount         used: 5.0 mg; MW=242.734     -   b. ALD-5-batch 1 (Legacy ID: D3); amount used: 6.1 mg;         MW=203.24; structure:

-   -   c. ALD-2-batch 1 Legacy ID: D2); amount used: 5.6 mg; MW=203.24;         structure:

2. Formulation and Stock Solution Preparation

-   -   a. Dimethyl sulfoxide (DMSO) at 100% was used for all samples.     -   b. Observations:         -   i. ALD-6: (5 mg, 20.6 μmol) of ALD-6 was dissolved in 0.206             ml of DMSO for a 100 mM stock solution. The 100 mM             ALD-6/DMSO solution was clear. Log dilutions were done with             DPBS. Upon dilution from 1:10 to 10 mM with DPBS, the             solution became cloudy, but cleared after vortex mixing. In             general, the benchmark concentration goal for DMSO in             primary neuronal cultures is less than 0.1%, to avoid             pharmacological effects from the DMSO. Note that 0.3% DMSO             was used for the 300 μM test concentration of all samples.             No apparent toxicity was observed in the assays after the 5             hr test.         -   ii. ALD-5: The 100 mM stock solution of ALD-5 was prepared             by dissolving 6.1 mg (30 μmol) into 0.3 ml of DMSO. The             ALD-5/DMSO mixture was a clear yellow solution. Log             dilutions were done with DPBS. Upon dilution from 1:10 to 10             mM with DPBS, the solution remained a clear yellow solution.         -   iii. ALD-2: The 100 mM stock solution of ALD-2 was prepared             by dissolving 5.6 mg (27.55 μmol) into 0.275 ml of DMSO. The             ALD-2/DMSO mixture was a clear amber solution. Log dilutions             were done with DPBS. Upon dilution from 1:10 to 10 mM with             DPBS, the solution remained a clear amber solution.     -   c. Details on the preparation of stock solutions: Compound         dilution in DMSO/DPBS         -   i. Stock A was 100 mM of compound in 100% DMSO.         -   ii. Stock B was prepared by adding 50 μl of stock A into 450             μl of DPBS for a final concentration of 10 mM. Added 3.3 μl             into 100 μl DPBS to yield a final concentration of 300 μM in             0.3% DMSO.         -   iii. Stock C was prepared by adding 50 μl of stock B into             450 μl of DPBS for a final concentration of 1 mM. Added 10             μM into 100 μl DPBS to yield a final concentration of 100 μM             in 0.1% DMSO.         -   iv. Stock D was prepared by adding 50 μl of stock C into 450             μl of DPBS for a final concentration of 100 μM. Added 10 μl             into 100 μl DPBS to yield a final concentration of 10 μM in             0.01% DMSO.         -   v. Stock E was prepared by adding 50 μl of stock D into 450             μl of DPBS for a final concentration of 10 μM. Added 10 μl             into 100 μl DPBS to yield a final concentration of 1 μM in             0.001% DMSO.         -   vi. Stock F was prepared by adding 50 μl of stock E into 450             μl of DPBS for a final concentration of 1 μM. Added 10 μl             into 100 μl DPBS for a final concentration of 0.1 μM in             0.0001% DMSO.         -   vii. 10 μl of the appropriate dilution was added to 90 μl             for a total volume of 100 μl in the well.

3. Culture Conditions Designed to Detect Neuroprotection from Oxidative Stress Associated with Hydrogen Peroxide:

-   -   a. Rat hippocampal cultures were prepared as previously         described (Brenneman D E, Smith G R, Zhang Y, Du Y, Kondaveeti S         K, Zdilla M J, Reitz A B. (2012) J. Molecular Neuroscience,         47:368-379). Under these conditions, the cultures are at least         90% neuronal. The most abundant non-neuronal cells are         astrocytes. Rat E18 hippocampal tissue was purchased from Brain         Bits, LLC (Springfield IL). Tissue was stored in Hibernate E         medium for transport.     -   b. All cultures were prepared into a 96-well format at a plating         density of 10K cells per well. Cultures were treated between day         10 and day 21 after dissociation of E18 hippocampal tissue. For         these experiments, all plates were treated on day 13. In all         experiments, the hydrogen peroxide was added to the cultures 10         minutes after treatment with the test agent or positive control         (cannabidiol). For each treatment condition, the number of         replicates was five.     -   c. All cultures were plated in B27/Neural Basal Medium. On the         day of treatment, all cultures were given a complete change of         medium into serum-free B27/Neural Basal Medium without         antioxidants.     -   d. As previously determined (Brenneman et al., 2012), 10 μM         hydrogen peroxide was used to produce toxicity and oxidative         stress. As described previously (Jarrett, S G, Liang, L-P,         Hellier, J L, Staley, K J and Patel, M. (2008) Neurobiol. Dis         30(1): 130-138) 10 μM hydrogen peroxide has been observed in the         hippocampus of rats with a kainate model of status epilepticus.     -   e. The positive control used in all studies was 10 μM         cannabidiol, a known antioxidant agent (Hampson et al. (1998),         Proc. Nat. Acad. Sci. 95:8268-8273) that is protective against         oxidative stress in primary neurons (Brenneman, D E, Petkanas, D         and Kinney, W. A. (2014) Annual Symposium on the Cannabinoids,         page 129).     -   f. Neither the negative control wells, the hydrogen peroxide         wells, nor the positive control wells contained any drug         vehicle.

4. Assays:

Both assays used in this study have been described in detail (Brenneman D E, Smith G R, Zhang Y, Du Y, Kondaveeti S K, Zdilla M J, Reitz A B. (2012) J. Molecular Neuroscience, 47:368-379).

-   -   a. The CFDA neuronal viability assay: In this assay, the CFDA         dye is taken up by all live cells and cleaved by esterases to         release fluorescein. The neuronal specificity is achieved         because neurons cannot remove this dye, whereas efflux of the         dye from non-neuronal cells can occur over time. After washing         away the extracellular dye, the cultures were read in a         fluorimeter; intracellular dye intensity is proportional to the         live neuronal population. Original reference: Petroski, R E and         Geller H M, (1994) “Selective labeling of embryonic neurons         cultures on astrocyte monolayers with 5(6)-carboxyfluorescein         diacetate (CFDA),” J. Neurosci. Methods 52:23.32. The mean         control level for each experiment is shown as a long-dashed         reference line.     -   b. A cell death assay, using propidium iodide, was conducted         simultaneously with the CFDA assay in the same well. This dye is         excluded from live cells and binds to the DNA of dead cells. The         assay detects both necrotic and apoptotic cell death; it does         not distinguish between neuronal cell death and non-neuronal         cell death. See Sarafian T A, Kouyoumjian S, Tashkin D, Roth         M D. (2002) Tox. Letters. 133: 171-179. The mean control level         is shown as a red medium-dashed reference line.     -   c. Reagents used         -   i. Hydrogen Peroxide solution, 30 wt %; Sigma-Aldrich             (216736-100 ml, Lot MKBV382V)         -   ii. Dimethyl Sulfoxide; Sigma-Aldrich (472301-100 ml) Batch             21096 JK         -   iii. Propidium Iodide; Sigma-Aldrich (P4864-10 ml; 1 mg/ml             solution in water)         -   iv. CFDA [5(6)-Carboxyfluorescein Diacetate] Sigma-Aldrich             Product Number: 21879-100 mg-F         -   v. Cannabidiol solution, 10 mg/ml in ethanol; Sigma-Aldrich             Product Number: 90899-1 ml         -   vi. Dulbecco's Phosphate Buffered Saline (DPBS). Gibco             (14190-144) Lot 1165767

5. Data Analyses:

-   -   a. Data Acquisition: Data were stored on Advanced Neural         Dynamics computers for analyses. Data acquisition was performed         on Cytofluor Fluorimeter and transferred to Excel spreadsheet         for analysis with Sigma Plot 11.     -   b. Statistical Analysis: All data were statistically analyzed by         an Analysis of Variance with the Multiple Comparisons versus         Control Group (Holm-Sidak) method. Statistical significance was         taken at the P<0.05 level. In all cases, comparisons were made         to the negative control (10 μM hydrogen peroxide treatment).     -   c. Methodology for EC₅₀ determination:         -   i. A broad concentration range was chosen to screen the             compounds in an EC₅₀ potency analysis. A log-based             concentration series from 0.1 μM to 300 μM was used.         -   ii. A nonlinear regression analysis was used to determine             the equation of the line that best fits the data. (Four             parameter Logistic curve)         -   iii. Based on the Logistic equation used in the preceding             Example, the EC₅₀s for neuroprotection were calculated and             plotted by SigmaPlot 11 to determine the concentration             required to produce half-maximal responses for both assays.             Drop lines were used to show the axes intersections             determining the EC₅₀.

B. Summary of Protection Studies for Aldeyra Compounds in Rat Hippocampal Cultures

TABLE 21 Summary of Protection Studies Data No effect Com- Formu- Full Efficacy Concen- Assay pound lation Concentration* tration EC50 ± SE CFDA ALD-6 DMSO 100 μM   1 μM  6.8 ± 1.2 μM CFDA ALD-5 DMSO Inactive Inactive Inactive CFDA ALD-2 DMSO Inactive Inactive Inactive PI ALD-6 DMSO  10 μM 0.1 μM 0.32 ± 0.03 μM PI ALD-5 DMSO Inactive Inactive Inactive PI ALD-2 DMSO Inactive Inactive Inactive *Concentration of test agent showing assay response levels not significantly different from that of no treatment controls.

C. Graphical Analyses of Experimental Findings and Raw Data

1. Experiment 1: Dose response of ALD-6. Effect on neuronal viability after co-treatment with 10 μM hydrogen peroxide.

-   -   a. Formulation: DMSO     -   b. Assay: CFDA     -   c. Toxin: 10 μM hydrogen peroxide     -   d. Duration of treatment: 5 hours     -   e. Growth medium: B27/neurobasal medium without antioxidants     -   f. Culture matrix: poly-L-lysine     -   g. Conclusions: The EC₅₀ was observed at 6.8÷1.2 μM; full         efficacy relative to controls (CBD+HP and no-treatment control)         was observed at 100 μM ALD-6. ALD-6 is fully neuroprotective         against hydrogen peroxide toxicity at 100 μM in this assay.         Results are shown in Table 22 and FIGS. 36 and 37 .

TABLE 22 Dose Response Data for ALD-6 Showing Effect on Neuronal Viability after Co-Treatment with 10 μM Hydrogen Peroxide CBD + ALD6 + ALD6 + ALD6 + ALD6 + ALD6 + Statistical HP HP HP HP HP HP HP Analysis Control 10 μM 300 μM 100 μM 10 μM 1 μM 0.1 μM 10 μM 59551 67121 65778 61821 47737 43459 41244 31224 55935 52880 53184 50555 53184 40839 36732 32978 61821 55631 53184 57177 45333 33090 31224 43036 59247 64665 53184 52880 53184 43340 31224 34419 54201 67958 59551 63181 40839 29547 31547 30376 Mean 58151*   57221*   56976*   57123*   48055*   38055 34394 34407 Std Error 1363 1977 2522 2449 2368 2845 2007 2268 P value * <0.001 <0.001 <0.001 <0.001 <0.001 N.S. N.S. N.S. % of Control 100 ± 2 98 ± 3 98 ± 4 98 ± 4 83 ± 4 65 ± 5 59 ± 3 59 ± 4 *Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

2. Experiment 2: Dose response effect of ALD-6 on cell death after co-treatment with 10 μM hydrogen peroxide.

-   -   a. Formulation: DMSO     -   b. Assay: Propidium Iodide     -   c. Toxin: 10 μM hydrogen peroxide     -   d. Duration of treatment: 5 hours     -   e. Growth medium: B27/neurobasal medium without antioxidants     -   f. Culture matrix: poly-L-lysine     -   g. Conclusions: The EC₅₀ was observed at 0.32±0.03 μM; full         efficacy relative to controls (CBD+HP and no-treatment control)         was observed at 10 μM ALD-6. ALD-6 is fully neuroprotective         against hydrogen peroxide toxicity in this assay. These data         suggest that the protective effect against cell death is more         potent than that observed for neuronal viability. Results are         shown in Table 23 and FIGS. 38 and 39 .

TABLE 23 Dose Response Data for ALD-6 Showing Effect on Neuronal Viability after Co-Treatment with 10 μM Hydrogen Peroxide CBD + ALD6 + ALD6 + ALD6 + ALD6 + ALD6 + Statistical HP HP HP HP HP HP HP Analysis Control 10 μM 300 μM 100 μM 10 μM 1 μM 0.1 μM 10 μM 159 119 159 167 179 214 225 225 159 159 189 193 183 183 250 366 171 171 189 206 189 223 294 294 163 123 147 189 189 183 242 255 171 197 197 135 139 189 356 329 Mean 165*   154*   176*   178*   176*   198*   273 294 Std Error 3 15 10 12 9 8 24 25 P value * <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 N.S. N.S. % of Control 100 ± 2 93 ± 9 107 ± 6 108 ± 7 107 ± 5 120 ± 5 165 ± 15 178 ± 15 *Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

3. Experiment 3: Dose response to ALD-5 in DMSO. Effect on neuronal viability after co-treatment with 10 μM hydrogen peroxide.

-   -   a. Formulation: DMSO     -   b. Assay: CFDA     -   c. Toxin: 10 μM hydrogen peroxide     -   d. Duration of treatment: 5 hours     -   e. Growth medium: B27/neurobasal medium without antioxidants     -   f. Culture matrix: poly-L-lysine     -   g. Conclusions: There was no statistically significant         neuroprotective activity from ALD-5 against hydrogen peroxide         toxicity from 0.1 to 300 μM. Results are shown in Table 24 and         FIG. 40 .

TABLE 24 Dose Response Data for ALD-5 Showing Effect on Neuronal Viability after Co-Treatment with 10 μM Hydrogen Peroxide CBD + ALD5 + ALD5 + ALD5 + ALD5 + ALD5 + Statistical HP HP HP HP HP HP HP Analysis Control 10 μM 300 μM 100 μM 10 μM 1 μM 0.1 μM 10 μM 55631 58812 40839 41143 39126 42756 39043 34930 58508 51551 43036 43743 37102 35588 38739 41244 58508 58812 41143 32978 41379 34189 40839 41925 57357 55983 44028 36732 34115 38997 38739 33203 54068 62790 42201 33429 32978 35707 37350 35588 Mean 56814*   57590*   42249 37605 36940 37447 38942 37378 Std Error 865 1858 592 2119 1552 1544 558 1764 P value * <0.001 <0.001 N.S. N.S. N.S. N.S. N.S. N.S. % of Control 100 ± 2 101 ± 3 74 ± 1 66 ± 4 65 ± 3 66 ± 3 69 ± 1 66 ± 3 *Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

4. Experiment 4: Dose response to ALD-5. Effect on cell death after co-treatment with 10 μM hydrogen peroxide.

-   -   a. Formulation: DMSO     -   b. Assay: Propidium Iodide     -   c. Toxin: 10 μM hydrogen peroxide     -   d. Duration of treatment: 5 hours     -   e. Growth medium: B27/neurobasal medium without antioxidants     -   f. Culture matrix: poly-L-lysine     -   g. Conclusions: There was no statistically significant         protection from cell death from 0.1 to 300 μM ALD-5. Results are         shown in Table 25 and FIG. 41 .

TABLE 25 Dose Response Data for ALD-5 Showing Effect on Cell Death after Co-Treatment with 10 μM Hydrogen Peroxide CBD + ALD5 + ALD5 + ALD5 + ALD5 + ALD5 + Statistical HP HP HP HP HP HP HP Analysis Control 10 μM 300 μM 100 μM 10 μM 1 μM 0.1 μM 10 μM 159 147 294 294 225 294 294 258 123 164 293 255 294 294 294 294 159 164 352 250 294 294 366 255 179 83 294 250 294 285 225 330 127 123 294 294 320 294 325 294 Mean 149* 136* 305 269 285 292 301 286 Std Error 11 15 12 10 16 2 23 14 P value * <0.001 <0.001 N.S. N.S. N.S. N.S. N.S. N.S. % of 100 ± 7 91 ± 10 205 ± 8 181 ± 7 191 ± 11 196 ± 1 202 ± 15 192 ± 9 Control *Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

5. Experiment 5: Dose response to ALD-2. Effect on neuronal viability after co-treatment with 10 μM hydrogen peroxide.

-   -   a. Formulation: DMSO     -   b. Assay: CFDA     -   c. Toxin: 10 μM hydrogen peroxide     -   d. Duration of treatment: 5 hours     -   e. Growth medium: B27/neurobasal medium without antioxidants     -   f. Culture matrix: poly-L-lysine     -   g. Conclusions: ALD-2 had no statistically significant         neuroprotection from decreases in neuronal viability in hydrogen         peroxide-treated cultures. Results are shown in Table 26 and         FIG. 42 .

TABLE 26 Dose Response Data for ALD-2 Showing Effect on Neuronal Viability after Co-Treatment with 10 μM Hydrogen Peroxide CBD + ALD2 + ALD2 + ALD2 + ALD2 + ALD2 + Statistical HP HP HP HP HP HP HP Analysis Control 10 μM 300 μM 100 μM 10 μM 1 μM 0.1 μM 10 μM 58877 55631 43036 45628 43601 43743 40839 34813 58508 54240 45924 35522 38997 36732 33885 40839 56160 50251 45628 36732 50251 43036 40839 43036 55631 62291 36732 43176 36732 35522 40839 31224 55807 52880 35048 32311 32978 31331 34813 41244 Mean 56997*   55059*   41274 38674 40512 38073 38243 38231 Std Error 700 2015 2270 2478 2981 2351 1596 2233 P value * <0.001 <0.001 N.S. N.S. N.S. N.S. N.S. N.S. % of 100 ± 1 97 ± 4 72 ± 4 68 ± 4 71 ± 5 67 ± 4 67 ± 3 67 ± 4 Control *Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

6. Experiment 6: Dose response to ALD-2. Effect on cell death after co-treatment with 10 μM hydrogen peroxide.

-   -   a. Formulation: DMSO     -   b. Assay: Propidium Iodide     -   c. Toxin: 10 μM hydrogen peroxide     -   d. Duration of treatment: 5 hours     -   e. Growth medium: B27/neurobasal medium without antioxidants     -   f. Culture matrix: poly-L-lysine     -   g. Conclusions: There was no statistically significant         protection from cell death produced by hydrogen peroxide after         treatment with ALD-2. Results are shown in Table 27 and FIG. 43         .

TABLE 27 Dose Response Data for ALD-2 Showing Effect on Cell Death after Co-Treatment with 10 μM Hydrogen Peroxide CBD + ALD2 + ALD2 + ALD2 + ALD2 + ALD2 + Statistical HP HP HP HP HP HP HP Analysis Control 10 μM 300 μM 100 μM 10 μM 1 μM 0.1 μM 10 μM 143 104 294 294 225 242 266 398 159 175 242 225 225 298 294 255 159 139 302 225 293 229 258 242 159 159 294 258 329 294 294 294 175 147 320 242 258 258 259 259 Mean 159*   145*   290 249 266 264 274 290 Std Error 5 12 13 13 20 14 8 28 P value * <0.001 <0.001 N.S. N.S. N.S. N.S. N.S. N.S. % of Control 100 ± 3 91 ± 8 182 ± 8 157 ± 8 167 ± 13 166 ± 9 172 ± 5 182 ± 18 *Significantly different from treatment with 10 μM hydrogen peroxide alone. N.S.: Not significantly different from treatment with 10 μM hydrogen peroxide alone.

7. Summary of Observations and Conclusions

-   -   a. ALD-6 exhibited neuroprotective activity against hydrogen         peroxide toxicity in the CFDA assay.     -   b. The neuroprotective effect of ALD-6 in the CFDA assay was not         statistically different from the control (no HP treatment) and         positive control (CBD) values, indicating full protection.     -   c. Full protection relative to controls was observed at 100 μM         ALD-6 in the CFDA neuronal viability assay. The no-effect         concentration for ALD-6 in the CFDA assay was 1 μM.     -   d. Non-linear curve fitting Logistic analyses indicated that the         EC₅₀ of ALD-6 in the CFDA neuronal viability assay was 6.8±1.2         μM.     -   e. ALD-6 exhibited protective activity from cell death from         hydrogen peroxide treatment in the Propidium Iodide (PI) assay.     -   f. The protective effect of ALD-6 from cell death in the PI         assay was not statistically different from that of the control         (no HP treatment) and positive control (CBD) values, indicating         full protection.     -   g. Full protection relative to no-treatment control values was         observed with ALD-6 at 10 μM in the PI assay, whereas the         no-effect concentration was 0.1 μM. ALD-6 response in the cell         death assay exhibited greater potency than in the CFDA assay. It         should be recognized that the cell death assay is not specific         to neurons and may involve non-neuronal cells that are present         in this model CNS system.     -   h. Non-linear Logistic curve fitting indicated that the EC₅₀ for         ALD-6 in the PI assay was 0.32±0.03 μM.     -   i. Treatment with ALD-5 or ALD-2 from 0.1 to 300 μM did not         produce statistically significant neuroprotection from hydrogen         peroxide treatment alone as assessed with CFDA assay.     -   j. Treatment with ALD-5 or ALD-2 from 0.1 to 300 μM did not         produce statistically significant protection from cell death         produced by hydrogen peroxide treatment as assessed with the PI         assay.     -   k. The toxic signal produced by 10 μM hydrogen peroxide was         typical of a wide variety of oxidative stressors (ethanol, heavy         metals, ammonium acetate, and glutamate) that have been tested         in the past, with decreases from control ranging from 30 to 50%.     -   l. The positive control (10 μM cannabidiol) was active on every         test plate, indicating that the model system was responding in a         typical protective manner.

Example 14: In Vivo Pharmacology of NS2-D6 (Compound I-1)

NS2-D6 (Compound ID 100029054-1; Batch Number 1603356191) was tested in binding and enzyme uptake assays. Compound binding was calculated as a % inhibition of the binding of a radioactively labeled ligand specific for each target. Compound enzyme inhibition effect was calculated as a % inhibition of control enzyme activity. Results showing an inhibition or stimulation higher than 50% are considered to represent significant effects of the test compounds. Such effects were observed here and are listed in the following tables.

Reference Compounds

In each experiment and if applicable, the respective reference compound was tested concurrently with NS2-D6, and the data were compared with historical values determined at the same research facility. The experiment was conducted in accordance with industry standard operating procedures.

Results

Table 28 summarizes the enzyme inhibition results.

TABLE 28 Summary of Enzyme Inhibition Results Assay 1.0E−05M 5-HT2B^((h)) (agonist radioligand) 62.5% acetylcholinesterase (h) 52.9% MAO-A (antagonist radioligand) 68.5% MT₃ (ML₂) (agonist radioligand) 71.1% PR (h) (agonist radioligand) 60.9%

Test compound results are shown in FIGS. 44-46 . Table 29 shows specific binding results for NS2-D6.

TABLE 29 Test Compound Results Client Compound Test % Inhibition of Control Specific Binding Compound I.D. I.D. Concentration 1^(st) 2^(nd) Mean A₁(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 3.5 5.3 4.4 A_(2A (h)) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −11.2 −15.0 −13.1 A_(2B)(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 13.2 4.3 8.7 A₃(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 17.3 15.1 16.2 α_(1A)(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 6.1 −3.3 1.4 α_(1B)(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −1.8 1.0 −0.4 α_(2A)(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 3.2 10.0 6.6 α_(2B)(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 15.9 20.8 18.4 α_(2C)(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −8.0 6.2 −0.9 β₁(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 9.7 11.0 10.3 β₂(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −0.6 5.8 2.6 Adrenergic beta3 100029054-1 NS2-d6 1.0E−05M 16.2 3.0 9.6 AT₁(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 10.7 −3.3 3.7 AT₂(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −3.5 −5.6 −4.6 APJ (apelin)(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 0.8 2.2 1.5 BZD (central) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −17.2 −16.6 −16.9 BB₃(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −17.1 4.6 −6.3 B₂(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −4.5 0.1 −2.2 CB₁(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 21.9 20.7 21.3 CB₂(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 16.6 5.0 10.8 CCK₁ (CCKA) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −10.0 11.9 0.9 CCK₂ (CCKB) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −3.6 −14.6 −9.1 CRF₁(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 6.7 5.7 6.2 D₁(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 0.8 6.6 3.7 D_(2S)(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 15.2 14.4 14.8 D₃(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 6.2 −4.1 1.1 ET_(A)(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −0.8 −5.8 −3.3 ET_(B)(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 7.6 −5.4 1.1 GAB_(A1)(h) (α1 β2, y2)(agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −45.9 −21.1 −33.5 GABA_(B(1b))(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −7.2 5.2 −1.0 glucagon(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −4.2 −6.9 −5.6 AMPA (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −5.9 5.1 −0.4 kainate (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 17.5 12.1 14.8 NMDA (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 5.5 14.6 10.1 glycine (strychnine-insenstive) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −7.7 16.2 4.2 TNF-α (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −6.5 −0.9 −3.7 CCR2 (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −12.4 6.2 −3.1 H₁(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −8.6 19.9 5.6 H₂(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 1.1 −0.5 0.3 H₃(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 15.3 13.9 14.6 H₄(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 9.8 −3.3 3.2 BLT₁ (LTB₄₎(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 9.6 7.0 8.3 CysLT₁(LTD₄₎(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −3.6 −17.1 −10.4 MCH₁ (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −3.7 −9.3 −6.5 MC₁ (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −10.5 −5.7 −8.1 MC₃ (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −1.4 0.6 −0.4 MC₄ (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −2.8 −3.0 −2.9 MT₁(ML_(1A))(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 30.0 37.7 33.9 MT₃ (ML₂)(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 69.0 73.3 71.1 MAO-A (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 66.4 70.7 68.5 motilin (h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −12.2 −3.9 −8.0 M₁ (h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −2.6 0.5 −1.1 M₂ (h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −4.2 −0.4 −2.3 M₃ (h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 4.6 13.6 9.1 M₄ (h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −1.0 9.2 4.1 NK₁ (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 4.2 4.1 4.2 NK₂ (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 18.6 −4.5 7.0 Y₁ (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −8.7 7.5 −0.6 N neuronal α4β2 (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −5.7 −6.7 −6.2 N muscle-type (h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −16.8 0.7 −8.1 δ (DOP)(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 3.8 −1.4 1.2 κ (KOP) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 13.3 16.2 14.8 μ (MOP) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 3.0 6.8 4.9 NOP (ORLI) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 10.8 18.7 14.8 PPARy (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 13.1 23.6 18.3 PAF (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −10.1 7.4 −1.3 PCP (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 2.1 6.5 4.3 EP₂(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 18.1 41.5 29.8 FP (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 10.7 −4.5 3.1 IP (PGI₂) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 5.0 −9.1 −2.0 LXRβ (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −10.5 −3.9 −7.2 5-HT_(1A) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 5.4 −1.8 1.8 5-HT_(1B) (h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −16.3 0.5 −7.9 5-HT_(1D) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −3.4 −5.1 −4.2 5-HT_(2A) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 5.4 17.8 11.6 5-HT_(2B) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 60.8 64.2 62.5 5-HT_(2C) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −3.1 10.0 3.4 5-HT₃ (h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −3.0 2.2 −0.4 5-HT_(4e) (h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −5.5 −2.6 −4.1 5-HT_(5a) (h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 3.8 17.3 10.6 5-HT₆ (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 1.1 17.7 9.4 5-HT₇ (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 2.3 −5.6 −1.7 sigma (non-selective) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 16.3 13.5 14.9 sst₁ (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −5.1 5.0 −0.1 sst₄ (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 3.1 −5.9 −1.4 GR (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M −6.7 4.1 −1.3 Estrogen ER alpha (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 15.8 4.3 10.1 PR (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 58.7 63.1 60.9 AR (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 41.4 44.3 42.8 Thyroid Hormone 100029054-1 NS2-d6 1.0E−05M −7.9 −5.9 −6.9 UT(h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 3.1 4.9 4.0 VPAC₁ (VIP₁) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 0.2 1.9 1.1 V_(1a) (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 2.1 −1.2 0.5 V₂ (h) (agonist radioligand) 100029054-1 NS2-d6 1.0E−05M 0.4 2.9 1.7 Ca²⁺ channel (L. dihydropyridine site) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 5.9 3.8 4.9 Ca²⁺ channel (L. diltiazem site) (benzothiazepines) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 14.8 13.3 14.0 Ca²⁺ channel (L. verapamil site) (phenylalkylamine) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −6.5 −11.8 −9.1 Ca²⁺ channel (N) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 4.6 8.1 6.3 Potassium Channel hERG (human)- [3H] Dofetilide 100029054-1 NS2-d6 1.0E−05M 8.0 −2.8 2.6 SK_(Ca) channel (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −10.8 −4.5 −7.7 Na⁺ channel (site 2) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −2.7 6.2 1.8 Cl⁻ channel (GABA-gated) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 33.9 42.6 38.2 norepinephrine transporter (h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 33.9 29.1 31.5 dopamine transporter (h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 19.9 27.3 23.6 GABA transporter (h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −6.1 −4.5 −5.3 choline transporter (CHT1)(h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M −17.9 −13.1 −15.5 5-HT transporter (h) (antagonist radioligand) 100029054-1 NS2-d6 1.0E−05M 2.9 5.2 4.0

Table 30 shows IC₅₀, K_(i), and nH values for various reference compounds that were compared with NS2-D6.

TABLE 30 Reference Compound Results Compound I.D. IC₅₀ (M) K_(i) (M) nH A₁ (h)(agonist radioligand) CPA 3.3E−09M 1.3E−09M 1.2 A_(2A) (h) (agonist radioligand) NECA 3.7E−08M 3.1E−08M 0.8 A_(2B) (h) (antagonist radioligand) NECA 5.5E−07M 5.1E−07M 0.9 A₃ (h) (agonist radioligand) IB-MECA 2.8E−10M 1.6E−10M 0.8 α_(1A) (h) (antagonist radioligand) WS 4101 2.5E−10M 1.2E−10M 1.1 α_(1B) (h) (antagonist radioligand) prazosin 2.5E−10M 6.7E−11M 1.2 α_(2A) (h) (antagonist radioligand) yohimbine 7.5E−09M 3.3E−09M 1.3 α_(2B) (h) (antagonist radioligand) yohimbine 7.6E−09M 5.0E−09M 1.0 α_(2C) (h) (antagonist radioligand) yohimbine 4.3E−09M 1.4E−09M 1.3 β1 (h) (agonist radioligand) atenolol 3.1E−07M 1.7E−07M 0.9 β2 (h) (agonist radioligand) ICI 118551 1.3E−09M 4.5E−10M 1.8 Adrenergic beta3 Alprenolo 1 1.4E−07M 1.0E−07M 0.6 AT₁ (h) (antagonist radioligand) saralasin 1.4E−09M 6.9E−10M 1.1 AT₂ (h) (agonist radioligand) angiotensin-II 1.2E−10M 5.9E−11M 0.8 APJ (apelin) (h) (agonist radioligand) apelin-13.TFA 2.7E−10M 2.5E−10M 1.0 BZD (central) (agonist radioligand) diazepam 7.4E−09M 6.2E−09M 0.9 BB₃ (h) (agonist radioligand) Bn(6-14) 9.8E−09M 6.0E−09M 1.0 B₂ (h) (agonist radioligand) NPC 567 2.4E−08M 1.2E−08M 1.0 CB₁ (h) (agonist radioligand) CP 55940 1.1E−09M 9.5E−10M 1.2 CB2 (h) (agonist radioligand) WIN 55212-2 2.1E−09M 1.4E−09M 1.0 CCK₁ (CCK_(A)) (h)(agonist radioligand) CCK-8s 1.1E−10M 8.3E−11M 0.9 CCK₂(CCK_(B)) (h)(agonist radioligand) CCK-8s 1.1E−10M 4.6E−11M 1.0 CRF₁ (h) (agonist radioligand) sauvagine 5.5E−10M 3.4E−10M 1.0 D₁ (h) (antagonist radioligand) SCH 23390 2.1E−10M 8.4E−11M 0.7 D_(2S) (h) (agonist radioligand) 7-OH-DPAT 1.2E−09M 4.8E−10M 0.7 D₃(h) (antagonist radioligand) (*) butaclamol 1.5E−09M 3.2E−10M 1.0 ET_(A) (h) (agonist radioligand) endothelin-1 5.4E−11M 2.7E−11M 1.0 ET_(B) (h) (agonist radioligand) endothelin-3 2.5E−11M 1.4E−11M 0.9 GABA_(A1) (h) (α1,(β2, γ2) (agonist radioligand) muscimol 7.0E−08M 4.7E−08M 0.8 GABA_(B(1b)) (h) (antagonist radioligand) CGP 54626 2.9E−09M 1.5E−09M 0.7 glucagon (h) (agonist radioligand) glucagon 1.5E−09M 1.1E−09M 0.6 AMPA (agonist radioligand) L-glutamate 2.8E−07M 2.6E−07M 0.9 kainate (agonist radioligand) kainic acid 2.3E−08M 1.8E−08M 0.9 NMDA (antagonist radioligand) CGS 19755 2.1E−07M 1.7E−07M 0.9 glycine (strychnine-insensitive) (antagonist radioligand) glycine 1.4E−07M 1.3E−07M 1.0 TNF-α (h) (agonist radioligand) TNF-alpha 6.9E−11M 2.3E−11M 1.3 CCR2 (h) (agonist radioligand) MCP-1 2.9E−11M 1.2E−11M 1.6 H₁ (h) (antagonist radioligand) pyrilamine 2.5E−09M 1.6E−09M 1.3 H₂ (h) (antagonist radioligand) cimetidine 6.5E−07M 6.3E−07M 0.8 H₃ (h) (agonist radioligand) (R)a-Me-histamine 1.9E−09M 4.7E−10M 1.4 H₄ (h) (agonist radioligand) imetit 4.4E−09M 1.9E−09M 0.9 BLT₁ (LTD₄) (h) (agonist radioligand) LTB₄ 4.2E−10M 2.1E−10M 0.8 CysLT₁ (LTD₄) (h) (agonist radioligand) LTD₄ 7.0E−10M 3.1E−10M 1.0 MCH₁ (h) (agonist radioligand) human MCH 4.9E−11M 4.5E−11M 1.0 MCI (agonist radioligand) NDP-α-MSH 1.8E−10M 8.9E−11M 1.0 MC3 (h) (agonist radioligand) NDP-α-MSH 2.0E−10M 1.7E−10M 1.2 MC₁ (h) (agonist radioligand) NDP-α-MSH 4.8E−10M 4.4E−10M 0.7 MT₁ (ML_(1A)) (h) (agonist radioligand) melatonin 2.0E−10M 1.6E−10M 1.6 MT₃ (ML₂) (agonist radioligand) melatonin 7.2E−08M 7.1E−08M 0.8 MAO-A (antagonist radioligand) clorgyline 1.7E−09M 1.0E−09M 1.7 motilin (h) (agonist radioligand) [Nleu¹³]-motilin 2.0E−09M 1.7E−09M 1.1 M₁ (h) (antagonist radioligand) pirenzepine 2.2E−08M 1.9E−08M 1.0 M₂ (h) (antagonist radioligand) methoctramine 4.9E−08M 3.4E−08M 0.8 M₃ (h) (antagonist radioligand) 4-DAMP 1.8E−09M 1.3E−09M 1.3 M₄ (h) (antagonist radioligand) 4-DAMP 1.5E−09M 9.5E−10M 1.2 NK₁ (h) (agonist radioligand) (Sar⁹,Met(O₂)¹¹]-SP 4.9E−10M 2.2E−10M 1.6 NK₂ (h) (agonist radioligand) [Nleu¹⁰]-NKA (4-10) 3.1E−09M 1.7E−09M 0.8 Y₁ (h) (agonist radioligand) NPY 9.4E−11M 6.7E−11M 1.3 N neuronal α4β2 (h) (agonist radioligand) nicotine 4.5E−09M 1.5E−09M 0.9 N muscle-type (h) (antagonist radioligand) α-bungarotoxin 2.3E−09M 2.1E−09M 1.2 δ (DOP) (h) (agonist radioligand) DPDPE 2.7E−09M 1.6E−09M 0.9 κ (KOP) (agonist radioligand) U50488 9.3E−10M 6.2E−10M 1.0 μ (MOP) (h) (agonist radioligand) DAMGO 2.8E−10M 1.2E−10M 0.7 NOP (ORLI) (h) (agonist radioligand) nociceptin 8.4E−10M 1.1E−10M 1.1 PPAR_(γ) (h) (agonist radioligand) rosiglitazone 1.2E−08M 6.1E−09M 0.9 PAF (h) (agonist radioligand) C₁₆-PAF 5.8E−09M 2.9E−09M 1.8 PCP (antagonist radioligand) MK 801 9.2E−09M 5.2E−09M 1.3 EP₂ (h) (agonist radioligand) PGE2 3.4E−09M 1.7E−09M 1.1 FP (h) (agonist radioligand) PGF2alpha 1.9E−09M 1.2E−09M 0.9 IP (PGI₂) (h) (agonist radioligand) iloprost 1.8E−08M 1.0E−08M 0.9 LXRβ (h) (agonist radioligand) 22(R)-hydroxycholesterol 4.0E−06M 2.7E−06M 1.1 5-HT_(1A) (h) (agonist radioligand) 8-OH-DPAT 5.8E−10M 3.6E−10M 0.8 5-HT_(1B) (antagonist radioligand) serotonin 4.9E−09M 3.0E−09M 0.9 5-HT_(1D) (agonist radioligand) serotonin 2.4E−09M 8.1E−10M 1.2 5-HT_(2A) (h) (agonist radioligand) (±)DO1 3.4E−10M 2.5E−10M 0.7 5-HT_(2B) (h) (agonist radioligand) (±)DO1 6.9E−09M 3.4E−09M 0.9 5-HT_(2C) (h) (agonist radioligand) (±)DO1 5.3E−10M 4.7E−10M 1.1 5-HT₃ (h) (antagonist radioligand) MDL 72222 6.9E−09M 4.8E−09M 0.9 5-HT_(4e) (h) (antagonist radioligand) serotonin 2.7E−07M 8.9E−08M 0.7 5-HT_(5α) (h) (agonist radioligand) serotonin 1.5E−07M 7.5E−08M 1.0 5-HT₆ (h) (agonist radioligand) serotonin 2.0E−07M 9.3E−08M 1.1 5-HT₇ (h) (agonist radioligand) serotonin 3.4E−10M 1.3E−10M 1.0 sigma (non-selective) (h) (agonist radioligand) haloperidol 6.3E−08M 5.1E−08M 0.7 sst₁ (h) (agonist radioligand) somatostatin-28 2.0E−10M 1.9E−10M 0.8 sst₄ (h) (agonist radioligand) somatostatin-14 9.1E−10M 8.9E−10M 0.9 GR (h) (agonist radioligand) dexamethasone 4.9E−09M 2.4E−09M 1.2 Estrogen ER alpha (h) (agonist radioligand) Diethylstilbestrol 3.7E−10M 1.0E−10M 1.9 PR (h) (agonist radioligand) promegestone 4.7E−10M 3.8E−10M 1.6 AR (h) (agonist radioligand) mibolerone 1.6E−09M 6.9E−10M 1.2 Thyroid Hormone Triiodothyronine 4.2E−11M 2.3E−11M 1.1 UT (h) (agonist radioligand) urotensin-II 7.7E−10M 5.8E−10M 1.1 VPAC₁ (VIP₁) (h) (agonist radioligand) VIP 3.5E−10M 1.9E−10M 1.9 V_(1a) (h) (agonist radioligand) [d(CH₂)₅ ¹, Tyr(ME)₂]-AVP 1.4E−09M 8.8E−10M 1.0 V₂ (h) (agonist radioligand) AVP 4.3E−10M 3.1E−10M 0.7 Ca²⁺ channel (L. dihydropyridine site) (antagonist radioligand) nitrendipine 3.0E−10M 1.9E−10M 1.1 Ca²⁺ channel (L. diltiazem site) (benzothiazepines) (antagonist radioligand) diltiazem 6.8E−08M 5.3E−08M 1.1 Ca²⁺ channel (L. verapamil site) (phenylalkylamine) (antagonist radioligand) D 600 2.7E−08M 1.3E−08M 0.5 Ca²⁺ channel (N) (antagonist radioligand) ω-conotoxin GVIA 1.7E−12M 6.8E−13M 0.8 Potassium Channel hERG (human)- [3H] Dofetilide Terfenadine 3.4E−08M 2.3E−08M 0.8 SK_(Ca) channel (antagonist radioligand) apamin 8.8E−12M 4.4E−12M 1.0 Na⁺ channel (site 2) (antagonist radioligand) veratridine 3.9E−06M 3.5E−06M 0.8 Cl⁻ channel (GABA-gated) (antagonist radioligand) picrotoxinin 2.8E−07M 2.4E−07M 1.0 norepinephrine transporter (h) (antagonist radioligand) protriptyline 2.8E−09M 2.1E−09M 1.0 dopamine transporter (h) (antagonist radioligand) BTCP 9.5E−09M 5.0E−09M 1.1 GABA transporter (h) (antagonist radioligand) nipecotic acid 2.5E−06M 2.5E−06M 0.8 choline transporter (CHT1)(h) (antagonist radioligand) hemicholinium-3 7.5E−09M 4.2E−09M 1.0 5-HT transporter (h) (antagonist radioligand) imipramine 2.7E−09M 1.3E−09M 1.3

FIG. 47 shows a histogram of in vitro pharmacology results in enzyme and uptake assays for NS2-D6.

Table 31 shows % inhibition of control values for NS2-D6.

TABLE 31 Test Compound Results Client Test % inhibition of Control Values Flags Compound I.D. Compound I.D. Concentration 1^(st) 2^(nd) Mean 1^(st) 2^(nd) COX1(h) 100029054-1 NS2-d6 1.0E−05 M 20.1 −2.6 8.8 COX2(h) 100029054-1 NS2-d6 1.0E−05 M 12.8 3.0 7.9 5-lipoxygenase (h) 100029054-1 NS2-d6 1.0E−05 M 23.5 24.8 24.2 12-lipoxygenase (h) 100029054-1 NS2-d6 1.0E−05 M −9.4 −4.1 −6.7 inducible NOS 100029054-1 NS2-d6 1.0E−05 M 2.8 −2.0 0.4 PDE2A1 (h) 100029054-1 NS2-d6 1.0E−05 M 17.1 −24.5 −3.7 PDE3B (h) 100029054-1 NS2-d6 1.0E−05 M 0.9 −4.5 −1.8 PDE4D2 (h) 100029054-1 NS2-d6 1.0E−05 M 15.9 16.9 16.4 PDE5 (h) (non-selective) 100029054.1 NS2-d6 1.0E−05 M −8.6 −2.9 −5.7 PDE6 (non-selective) 100029054.1 NS2-d6 1.0E−05 M 4.1 18.9 11.5 ACE (h) 100029054.1 NS2-d6 1.0E−05 M 127.6 131.0 129.3 INTER INTER ACE-2 (h) 100029054-1 NS2-d6 1.0E−05 M 23.8 26.2 25.0 INTER INTER BACE-1 (h) (β-secretase) 100029054-1 NS2-d6 1.0E−05 M −4.1 −5.7 −4.9 INTER INTER caspase-3 (h) 100029054-1 NS2-d6 1.0E−05 M −2.0 2.5 0.2 caspase-8 (h) 100029054-1 NS2-d6 1.0E−05 M 14.7 16.4 15.6 HIV-1 protease 100029054-1 NS2-d6 1.0E−05 M 16.6 4.8 10.7 MMP-1 (h) 100029054-1 NS2-d6 1.0E−05 M 45.3 10.7 28.0 INTER INTER MMP-2 (h) 100029054-1 NS2-d6 1.0E−05 M 4.9 −2.4 1.3 MMP-9 (h) 100029054-1 NS2-d6 1.0E−05 M 4.5 4.4 4.4 INTER INTER Abl kinase (h) 100029054-1 NS2-d6 1.0E−05 M −4.8 −11.6 −8.2 CaMK2α (h) 100029054-1 NS2-d6 1.0E−05 M 0.2 −6.5 −3.1 CDK2 (h) (cycA) 100029054-1 NS2-d6 1.0E−05 M 1.2 −5.1 −1.9 ERK₂ (h) (P42^(mapk)) 100029054-1 NS2-d6 1.0E−05 M −6.3 −1.3 −3.8 FLT-1 kinase (h) (VEGFR1) 100029054-1 NS2-d6 1.0E−05 M −15.5 −18.8 −17.2 Fyn kinase (h) 100029054-1 NS2-d6 1.0E−05 M −8.6 −0.3 −4.5 IRK (h)(InsR) 100029054-1 NS2-d6 1.0E−05 M −15.1 −7.9 −11.5 Lyn A kinase (h) 100029054-1 NS2-d6 1.0E−05 M −17.7 −35.7 −26.7 p38α kinase (h) 100029054-1 NS2-d6 1.0E−05 M −3.2 −3.1 −3.2 ZAP70 kinase (h) 100029054-1 NS2-d6 1.0E−05 M 0.4 1.5 1.0 acetylcholinesterase (h) 100029054-1 NS2-d6 1.0E−05 M 45.8 60.1 52.9 COMT (catechol-O-methyl transferase) 100029054-1 NS2-d6 1.0E−05 M 5.8 14.7 10.3 INTER INTER MAO-B (h) recombinant enzyme 100029054-1 NS2-d6 1.0E−05 M −3.1 −3.7 −3.4 xanthine oxldase/superoxide 0₂ ⁻scavenging 100029054-1 NS2-d6 1.0E−05 M −3.1 15.6 6.3 ATPase (Na+/K+) 100029054-1 NS2-d6 1.0E−05 M 5.2 −0.4 2.4 Peptidase, Metalloproteinase, Neutral Endopeptidase 100029054-1 NS2-d6 1.0E−05 M 0.3 3.0 1.7 INTER: Test compound interferes with the assay detection method.

Table 32 shows IC₅₀ and nH values for reference compounds.

TABLE 32 Reference Compound IC₅₀ and nH Values Compound I.D. IC₅₀ (IV) nH COX1(h) Diclofenac 5.7E−09M 1.6 COX2(h) NS398 6.8E−08M 1.3 5-lipoxygenase (h) NDGA 2.3E−07M 1.6 12-lipoxygenase (h) NDGA 5.1E−07M 1.2 inducible NOS 1400W 4.0E−08M 1.4 PDE2A1 (h) EHNA 1.1E−06M 0.8 PDE3B (h) milrinone 1.0E−06M 0.9 PDE4D2 (h) Ro 20-1724 8.2E−07M 1.0 PDE5 (h) (non-selective) dipyridamole 1.8E−06M 1.1 PDE6 (non-selective) zaprinast 1.8E−07M 1.0 ACE (h) captopril 5.7E−10M 1.2 ACE-2 (h) Ac-GG-26-NH₂ 2.9E−07M 2.4 BACE-1 (h) ((β-secretase) OM 99-2 1.2E−07M 1.4 caspase-3 (h) Ac-DEVD-CHO 2.0E−09M 1.1 caspase-8 (h) Ac-lETD-CHO 2.8E−08M 0.8 HIV-1 protease pepstatin A 2.2E−06M 1.9 MMP-1 (h) GM6001 1.6E−09M 1.3 MMP-2 (h) GM6001 1.5E−09M 1.3 MMP-9 (h) GM6001 5.4E−10M 0.9 Abl kinase (h) staurosporine 2.6E−07M 1.4 CaMK2u (h) AIP 2.6E−07M 1.1 CDK2 (h) (cycA) staurosporine 6.9E−09M 1.0 ERK2 (0) (P42^(mapk)) staurosporine 6.9E−07M 1.0 FLT-1 kinase (h) (VEGFR1) staurosporine 7.0E−09M 0.7 Fyn kinase (h) PP1 1.0E−07M >3 IRK (h) (InsR) staurosporine 1.6E−08M 0.9 Lyn A kinase (h) staurosporine 1.2E−08M 1.9 p38α kinase (h) SB202190 3.0E−08M 1.0 ZAP70 kinase (h) staurosporine 1.1E−07M 1.4 acetylcholinesterase (h) galanthamine 7.6E−07M 1.0 COMT (catechol-O-methyl transferase) Ro 41-0960 3.0E−08M 1.7 MAO-B (h) recombinant enzyme deprenyl 3.5E−08M 1.4 xanthine oxidase/superoxide 0₂-scavenging allopurinol 2.0E−06M 1.3 ATPase (Na+/K+) ouabain 1.0E−06M 1.3 Peptidase, Metalloproteinase, Neutral Endopeptidase Phosphoramidon 1.6E−08M 0.9

Table 33 shows test compound results of NS2-D6 on guanylyl cyclase.

TABLE 33 Test Compound Results with Guanylyl Cyclase Client Test % of Control Values Compound I.D. Compound I.D. Concentration 1^(st) 2^(nd) Mean guanylyl cyclase (h) (activator effect) 100029054-1 NS2-d6 1.0E−05M 0.2 0.3 0.2

Table 34 shows reference compound EC₅₀ results on guanylyl cyclase.

TABLE 34 Reference Compound Results with Guanylyl Cyclase Compound I.D. EC₅₀(M) nH guanylyl cyclase (h) (activator effect) sodium nitroprusside 3.5E−06M 2.2

Results showing an inhibition (or stimulation for assays run in basal conditions) higher than 50% are considered to represent significant effects of the test compounds. 50% is the most common cut-off value for further investigation (determination of IC₅₀ or EC₅₀ values from concentration-response curves). Results showing an inhibition (or stimulation) between 25% and 50% are indicative of weak to moderate effects (in most assays, they should be confirmed by further testing as they are within a range where more inter-experimental variability can occur). Results showing an inhibition (or stimulation) lower than 25% are not considered significant and mostly attributable to variability of the signal around the control level.

Low to moderate negative values have no real meaning and are attributable to variability of the signal around the control level. High negative values (≥50%) that are sometimes obtained with high concentrations of test compounds are generally attributable to nonspecific effects of the test compounds in the assays. On rare occasions they could suggest an allosteric effect of the test compound.

Experimental Conditions

Table 35 summarizes binding assay conditions

TABLE 35 Binding Assay Conditions Detection Assay Source Ligand Conc. Kd Non Specific Incubation Method Bibl. A1(h) (agonist human [³H]CCPA     1 nM    0.7 nM CPA   60 min Scintillation 198 radioligand) recombinant (10 μM) RT counting (CHO cells) A_(2A) (h)(agonist human [³H]CGS 21680     6 nM     27 nM NECA  120 min Scintillation 141 radioligand) recombinant (10 μM) RT counting (HEK-293 cells) A_(2B)(h) (antagonist human [³H]CPX     5 nM     65 nM NECA   60 min Scintillation 229 radioligand) recombinant (100 μM) RT counting (HEK-293 cells) A₃(h) (antagonist human [¹²⁵]AB-MECA  0.15 nM   0.22 nM IB-MECA  120 min Scintillation 206 radioligand) recombinant (1 μM) RT counting (HEK-293 cells) α_(1A)(h) (antagonist human [³H]prazosin   0.1 nM    0.1 nM epinephrine   60 min Scintillation 897 radioligand) recombinant (0.1 μM) RT counting (CHO cells) α_(1B)(h) (antagonist human [³H]prazosin  0.15 nM  0.055 nM phentolamine   60 min Scintillation 701 radioligand) recombinant (10 μM) RT counting (CHO cells) α_(2A)(h) (antagonist human [³H]RX 821002     1 nM    0.8 nM (−)epinephrine   60 min Scintillation 542 radioligand) recombinant (100 μM) RT counting (CHO cells) α_(2B)(h) (antagonist human [³H]RX 821002   2.5 nM      5 nM (−)epinephrine   60 min Scintillation 56 radioligand) recombinant (100 μM) RT counting (CHO cells) α_(2C)(h) (antagonist human [³H]RX 821002     2 nM   0.95 nM (−)epinephrine   60 min Scintillation 56 radioligand) recombinant (100 μM) RT counting (CHO cells) β₁(h) (agonist human [³H](−)CGP   0.3 nM   0.39 nM alprenolol   60 min Scintillation 548 radioligand) recombinant 12177 (50 μM) RT counting (HEK-293 cells) β₂(h) (agonist human [³H](−)CGP   0.3 nM   0.15 nM alprenolol  120 min Scintillation 794 radioligand) recombinant 12177 (50 μM) RT counting (CHO cells) Adrenergic beta3 human [125I]   0.5 nM    1.5 nM Alprenolol(100   90 min Scintillation 1277 recombinant Cyanopindolol (0.0 μM) 25° C. counting (HEK-293 cells) AT₁(h) (antagonist human [¹²⁵I][Sar¹, Ile⁸]-AT-  0.05 nM   0.05 nM angiotensin-II  120 min Scintillation 776 radioligand) recombinant II (10 μM) 37° C. counting (HEK-293 cells) AT₂(h) (agonist human [¹²⁵I]GCP  0.01 nM   0.01 nM angiotensin-II  4 hr Scintillation 248 radioligand) recombinant 42112A (1 μM) 37° C. counting (HEK-293 cells) APJ (apelin)(h) human [¹²⁵I]  0.03 nM   0.06 nM apelin-13  120 min Scintillation 846 (agonist radioligand) recombinant [Glpr⁶⁵, Nle⁷⁵], Tyr⁷⁷- (1 μM) RT counting (CHO cells) apelin-13 BB₃(h) (agonist human [¹²⁵I]Bn(6-14)  0.01 nM   0.16 nM Bn (6-14)   60 min Scintillation 287 radioligand) recombinant (1 μM) RT counting (CHO cells) B₂(h) (agonist human [³H]bradykinin   0.3 nM   0.32 nM bradykinin   60 min Scintillation 346 radioligand) recombinant (1 μM) RT counting (CHO cells) CB₁(h) (agonist human [³H](−)CP 55940   0.5 nM    3.5 nM WIN 55212-2  120 min Scintillation 857 radioligand) recombinant (10 μM) 37° C. counting (CHO cells) CB₂(h) (agonist human [³H]WIN   0.8 nM    1.5 nM WIN 55212-2  120 min Scintillation 165 radioligand) recombinant 55212-2 (5 μM) 37° C. counting (CHO cells) CCK₁ (CCK_(A)) (h) human [¹²⁵I]CCK-8s  0.08 nM   0.24 nM CCK-8s   60 min Scintillation 562 (agonist radioligand) recombinant (1 μM) RT counting (CHO cells) CCK₂ (CCK_(B)) (h) human [¹²⁵I]CCK-8s  0.08 nM  0.054 nM CCK-8s   60 min Scintillation 134 (agonist radioligand) recombinant (1 μM) RT counting (CHO cells) CRF₁(h) (agonist human [¹²⁵I]sauvagine 0.075 nM   0.12 nM sauvagine  120 min Scintillation 557 radioligand) recombinant (0.5 μM) RT counting (CHO cells) D₁(h) (antagonist human [³H]SCH 23390   0.3 nM    0.2 nM SCH 23390   60 min Scintillation 281 radioligand) recombinant (1 μM) RT counting (CHO cells) D_(2S)(h) (agonist human [³H]7-OH-DPAT     1 nM   0.68 nM butaclamol   60 min Scintillation 87 radioligand) recombinant (10 μM) RT counting (HEK-293 cells) D₃(h) (antagonist human [³H]methyl-   0.3 nM  0.085 nM (+)butaclamol   60 min Scintillation 145 radioligand) recombinant spiperone (10 μM) RT counting (CHO cells) ET_(A)(h) (agonist human [¹²⁵I]endothelin-1  0.03 nM   0.03 nM endothelin-1  120 min Scintillation 30 radioligand) recombinant (100 μM) 37° C. counting (CHO cells) ET_(B)(h) (agonist human [¹²⁵I]endothelin-1  0.03 nM   0.04 nM endothelin-1  120 min Scintillation 541 radioligand) recombinant (0.1 μM) 37° C. counting (CHO cells) GAB_(A1)(h) (α1 β2, human [³H]muscimol    15 nM     30 nM muscimol  120 min Scintillation 109 y2)(agonist recombinant (10 μM) RT counting radioligand) (CHO cells) GABA_(B(1b))(h) human [³H]CGP     1 nM      1 nM CGP 52432  120 min Scintillation 508 (antagonist recombinant 54626 (100 μM) RT counting radioligand) (CHO cells) glucagon(h) (agonist human [¹²⁵I]glucagon 0.025 nM  0.069 nM glucagon  120 min Scintillation 624 radioligand) recombinant (1 μM) RT counting (CHO cells) TNF-α (h) (agonist U-937 cells [¹²⁵I]TNF-α   0.1 nM   0.05 nM TNF-α  120 min Scintillation 26 radioligand) (10 μM)  4° C. counting CCR2 (h) (agonist human [¹²⁵I]MCP-1  0.01 nM  0.007 nM MCP-1   60 min Scintillation 13 radioligand) recombinant (10 nM) RT counting (HEK-293 cells) H₁(h) (antagonist human [³H]pyrilamine     1 nM    1.7 nM pyrilamine   60 min Scintillation 492 radioligand) recombinant (1 μM) RT counting (HEK-293 cells) H₂(h) (antagonist human [¹²⁵I]APT 0.075 nM    2.9 nM tiotidine  120 min Scintillation 540 radioligand) recombinant (100 μM) RT counting (CHO cells) H₃(h) (agonist human [³H]N^(α)-Me-     1 nM   0.32 nM (R)α-Me-   60 min Scintillation 563 radioligand) recombinant histamine histamine RT counting (CHO cells) (1 μM) H₄(h) (agonist human [³H]histamine    10 nM    7.6 nM imetit   60 min Scintillation 631 radioligand) recombinant (1 μM) RT counting (HEK-293 cells) BLT₁ (LTB₄₎(h) human [³H]LTB₄   0.2 nM    0.2 nM LTB₄   60 min Scintillation 616 (agonist radioligand) recombinant (0.2 μM) RT counting (CHO cells) CysLT₁ (LTD₄)(h) human [³H]LTD₄   0.3 nM   0.24 nM LTD₄   60 min Scintillation 618 (agonist radioligand) recombinant (1 μM) RT counting (CHO cells) MCH₁ (h) (agonist human [¹²⁵I]Phe¹³, Tyr¹⁹-   0.1 nM      1 nM human MCH   60 min Scintillation 526 radioligand) recombinant MCH (0.1 μM) RT counting (CHO cells) MC₁ (agonist B-16-F1 cells [¹²⁵I]NDP-α-MSH  0.05 nM   0.05 nM NDP-α-MSH   90 min Scintillation 390 radioligand) (endogenous) (1 μM) RT counting MC₃ (h) (agonist human [¹²⁵I]NDP-α-MSH 0.075 nM    0.4 nM NDP-α-MSH   60 min Scintillation 211 radioligand) recombinant (1 μM) 37° C. counting (CHO cells) MC₄ (h) (agonist human [¹²⁵I]NDP-α-MSH  0.05 nM   0.54 nM NDP-α-MSH  120 min Scintillation 211 radioligand) recombinant (1 μM) 37° C. counting (CHO cells) MT₁ (ML_(1A))(h) human [¹²⁵I]2-  0.01 nM   0.04 nM melatonin   60 min Scintillation 639 (agonist radioligand) recombinant iodomelatonin (1 μM) RT counting (CHO cells) MT₃ (ML₂)(h) Hamster brain [¹²⁵I]2-   0.1 nM    4.8 nM melatonin   60 min Scintillation 186 (agonist radioligand) iodomelatonin (30 μM)  4° C. counting motillin (h) human [¹²⁵I]motilin  0.05 nM   0.26 nM [Nleu¹³]-motilin  120 min Scintillation 285 (antagonist recombinant (1 μM) RT counting radioligand) (CHO cells) M₁ (h) (antagonist human [³H]pirenzepine     2 nM     13 nM atropine   60 min Scintillation 59 radioligand) recombinant (1 μM) RT counting (CHO cells) M₂ (h) (antagonist human [³H]AF-DX 384     2 nM    4.6 nM atropine   60 min Scintillation 59 radioligand) recombinant (1 μM) RT counting (CHO cells) M₃ (h) (antagonist human [³H]4-DAMP   0.2 nM    0.5 nM atropine   60 min Scintillation 546 radioligand) recombinant (1 μM) RT counting (CHO cells) M₄ (h) (antagonist human [³H]4-DAMP   0.2 nM   0.32 nM atropine   60 min Scintillation 59 radioligand) recombinant (1 μM) RT counting (CHO cells) NK₁ (h) (agonist U373MG [¹²⁵I]-Substance P  0.05 nM   0.04 nM [Sar⁹,   30 min Scintillation 104 radioligand) uppsala LYS3 Met(O₂)¹¹]-SP RT counting (1 μM) NK₂ (h) (agonist human [¹²⁵I]NKA   0.1 nM   0.12 nM [Nieu ¹⁰]-NKA   60 min Scintillation 3 radioligand) recombinant (4-10) RT counting (CHO cells) (300 μM) Y₁ (h) (agonist SK-N-MC [¹²⁵I]peptide YY 0.025 nM   0.06 nM NPY  120 min Scintillation 391 radioligand) cells (1 μM) 37° C. counting (endogenous) N neuronal α4β2 (h) SH-SY5Y [³H]cytisine   0.6 nM    0.3 nM nicotine  120 min Scintillation 1084 (agonist radioligand) cells (human (10 μM)  4° C. counting recombinant) N muscle-type (h) TE671 cells [¹²⁵I]α-   0.5 nM      5 nM a-bungarotoxin  120 min Scintillation 524 (antagonist (endogenous) bungarotoxin (5 μM) RT counting radioligand) ō (DOP) (h) (agonist human [³H]DADLE   0.5 nM   0.73 nM naltrexone  120 min Scintillation 501 radioligand) recombinant (10 μM) RT counting (CHO cells) κ (KOP) (h) (agonist rat [³H]U 69593     1 nM      2 nM naloxone   60 min Scintillation 771 radioligand) recombinant (10 μM) RT counting (CHO cells) μ (MOP) (h) (agonist human [³H]DAMGO   0.5 nM   0.35 nM naloxone  120 min Scintillation 260 radioligand) recombinant (10 μM) RT counting (HEK-293 cells) NOP (ORL1) (h) human [³H]nociceptin   0.2 nM    0.4 nM nociceptin   60 min Scintillation 7 (agonist radioligand) recombinant (1 μM) RT counting (HEK-293 cells) PPARy (h) (agonist human [³H]msiglhazone     5 nM    5.7 nM rosiglltazone  120 min Scintillation 567 radioligand) recombinant (10 μM)  4° C. counting (E. coli) PAF (h) (agonist human [³H]C₁₆-PAF   1.5 nM    1.5 nM WEB 2086   60 min Scintillation 531 radioligand) recombinant (10 μM) RT counting (CHO cells) EP₂ (h) (agonist human [³H]PGE₂     3 nM      3 nM PGE2  120 min Scintillation 781 radioligand) recombinant (10 μM) RT counting (HEK-293 cells) FP(h) (agonist human [³H]PGF_(2 α)     2 nM   3.83 nM cloprostenol (10   60 min Scintillation 781 radioligand) recombinant μM) RT counting (HEK-293 cells) IP(PGI₂)(h) (agonist human [³H]iloprost     6 nM      8 nM iloprost   60 min Scintillation 781 radioligand) recombinant (10 μM) RT counting (HEK-293 cells) LXRβ(h) (agonist human [³H]hydroxycholesterol    25 nM     55 nM 22(R)-   60 min Scintillation 856 radioligand) recombinant hydroxycholesterol RT counting (BL21/DE3 (30 μM) cells) 5-HT_(1A) (h) (agonist human [²H)8-OH-   0.3 nM    0.5 nM 8-OH-DPAT   60 min Scintillation 164 radioligand) recombinant DPAT (10 μM) RT counting (HEK-293 cells) 5-HT_(1B) (h) rat cerebral [¹²⁵I]CYP   0.1 nM   0.16 nM serotonin  120 min Scintillation 111 (antagonist codex (+30 μM (10 μM) 37° C. counting radioligand) isoproterenol) 5-HT_(1D) (h) (agonist rat (³H]serotonin     1 nM    0.5 nM serotonin   60 min Scintillation 777 radioligand) recombinant (10 μM) RT counting (CHO cells) 5-HT_(2A) (h) (agonist human [¹²⁵I](⁺)DOI   0.1 nM    0.2 nM (⁺)DOI   60 min Scintillation 288 radioligand) recombinant (1 μM) RT counting (HEK-293 cells) 5-HT_(2B) (h) (agonist human [¹²⁵I](⁺)DOI   0.2 nM    0.2 nM (⁺)DOI   60 min Scintillation 571 radioligand) recombinant (1 μM) RT counting (CHO cells) 5-HT_(2C) (h) (agonist human [¹²⁵I](⁺)DOI   0.1 nM    0.9 nM (⁺)DOI   60 min Scintillation 288 radioligand) recombinant (10 μM) 37° C. counting (HEK-293 cells) 5-HT_(4e) (h) human [³H]GR 113808   0.3 nM   0.15 nM serotonin   60 min Scintillation 309 (antagonist recombinant (100 μM) 37° C. counting radioligand) (CHO cells) 5-HT_(5a) (h) human [³H]LSD   1.5 nM    1.5 nM serotonin  120 min Scintillation 193 (antagonist recombinant (100 μM) 37° C. counting radioligand) (HEK-293 cells) 5-HT₆ (h) (agonist human [³H]LSD     2 nM    1.8 nM serotonin  120 min Scintillation 161 radioligand) recombinant (100 μM) 37° C. counting (CHO cells) 5-HT₇ (h) (agonist human [³H]LSD     4 nM    2.3 nM serotonin  120 min Scintillation 217 radioligand) recombinant (10 μM) RT counting (CHO cells) sigma (non-selective) Jurkat cells [3H]DTG    10 nM     41 nM Haloperidol (10  120 min Scintillation 1136 (h) (agonist (endogenous) μM) RT counting radioligand) sst₁ (h) (agonist human [¹²⁵I]Tyr¹¹-   0.1 nM      1 nM somatostatin-2  180 min Scintillation 761 radioligand) recombinant somatostatin-1 4 8 37° C. counting (CHO cells) (1 μM) sst₄ (h) (agonist human [¹²⁵I]Tyr¹¹-   0.1 nM    5.9 nM somatostatin-1  120 min Scintillation 296 radioligand) recombinant somatostatin-1 4 4 RT counting (CHO cells) (1 μM) GR (h) (agonist IM-9 cells [³H]dexamethasone   1.5 nM    1.5 nM triamcinolone  6 hr Scintillation 283 radioligand) (cytosol) (10 μM)  4° C. counting Estrogen ER alpha human [3H]Estradiol   0.5 nM   0.20 nM Diethylstilbestrol  120 min Scintillation 1280 (h) (agonist recombinant (1 μM) RT counting radioligand) (sf9 cells) PR (h) (agonist T47D cells [³H]progesterone   0.5 nM      2 nM promegestone (1 20 hr Scintillation 930 radioligand) (cytosol) μM)  4° C. counting AR (h) (agonist LNCaP cells [³H]methyltrienolone     1 nM    0.8 nM mibolerone (1 24 hr Scintillation 498 radioligand) (cytosol) μM)  4° C. counting Thyroid Hormone rat liver [125I]  0.03 nM  0.034 nM Triodothymnine 1080 min Scintillation 1289 Triiodothyronine (1.0 μM)  4° C. counting UT(h) (agonist human [¹²⁵I]urotensin-II   0.1 nM   0.29 nM urotensin-II (3  120 min Scintillation 622 radioligand) recombinant μM) RT counting (CHO cells) VPAC₁ (VIP₁)(h) human [¹²⁵I]VIP  0.04 nM   0.05 nM VIP   60 min Scintillation 50 (agonist radioligand) recombinant (1 μM) RT counting (CHO cells) V_(1a) (h) (agonist human [³H]AVP   0.3 nM    0.5 nM AVP   60 min Scintillation 343 radioligand) recombinant (1 μM) RT counting (CHO cells) V₂ (h) (agonist human [³H]AVP   0.3 nM   0.76 nM AVP 120 min Scintillation 343 radioligand) recombinant (1 μM) RT counting (CHO cells) Ion channels BZD (central) rat cerebral [³H]flunitrazepam   0.4 nM    2.1 nM diazepam   60 min Scintillation 227 (agonist radioligand) cortex (3 μM)  4° C. counting AMPA (agonist rat cerebral [³H]AMPA     8 nM     82 nM L-glutamate (1   60 min Scintillation 166 radioligand) cortex mM)  4° C. counting kainate (agonist rat cerebral [³H]kainic acid     5 nM     19 nM L-glutamate (1   60 min Scintillation 160 radioligand) cortex mM)  4° C. counting NMDA (antagonist rat cerebral [³H]CGP     5 nM     23 nM L-glutamate   60 min Scintillation 221 radioligand) cortex 39653 (100 μM)  4° C. counting glycine (strychnine- rat cerebral [³H]MDL   0.5 nM      5 nM glycine   45 min Scintillation 219 insenstive) cortex 105, 519 (1 mM)  0° C. counting (antagonist radioligand) PCP (antagonist rat cerebral [³H]TCP    10 nM     13 nM MK 801  120 min Scintillation 257 radioligand) cortex (10 μM) 37° C. counting 5-HT₃ (h) (antagonist human [³H]BRL 43694   0.5 nM   1.15 nM MDL 72222  120 min Scintillation 109 radioligand) recombinant (10 μM) RT counting (CHO cells) Ca²⁺ channel (L, rat cerebral [³H]initrendipine   0.1 nM   0.18 nM nitrendipine   90 min Scintillation 996 dihydropyridine site) cortex (1 μM) RT counting (antagonist radioligand) Ca²⁺ channel (L, rat cerebral [³H]diltiazem    15 nM     52 nM diltiazem  120 min Scintillation 212 diltiazem site) cortex (10 μM) RT counting (benzothiazepines) (antagonist radioligand) Ca²⁺ channel (L, rat cerebral [³H]D888     3 nM      3 nM D 600  120 min Scintillation 194 verapamil site) cortex (10 μM) RT counting (phenylalkylamine) (antagonist radioligand) Ca²⁺ channel (N) rat cerebral [¹²⁵I]ω- 0.001 nM 0.0007 nM w-conotoxin   30 min Scintillation 259 (antagonist cortex conotoxin GVIA RT counting radioligand) GVIA (10 μM) Potassium Channel human [³H]Dofetilide     3 nM    6.6 nM Terfenadine   60 min Scintillation 1398 hERG (human)-[3H] recombinant (25 μM) RT counting Dofetilide (HEK-293 cells) SK_(Ca) channel rat cerebral [¹²⁵I]apamin 0.007 nM  0.007 nM apamin   60 min Scintillation 112 (antagonist cortex (100 μM)  4° C. counting radioligand) Na⁺ channel (site 2) rat cerebral [³H]batrachotoxinin    10 nM     91 nM veratridine   60 min Scintillation 28 (antagonist cortex (300 μM) 37° C. counting radioligand) Cl⁻ channel (GABA- rat cerebral [³⁵S]TBPS     3 nM   14.6 nM picrotoxinin  120 min Scintillation 136 gated) (antagonist cortex (20 μM) RT counting radioligand) Transporters norepinephrine human [³H]nisoxetine     1 nM    2.9 nM desipramine 120 min Scintillation 190 transporter (h) recombinant (1 μM)  4° C. counting (antagonist (CHO cells) radioligand) dopamine human [³H]BTCP     4 nM    4.5 nM BTCP  120 min Scintillation 190 transporter (h) recombinant (10 μM)  4° C. counting (antagonist (CHO cells) radioligand) GABA transporter rat cerebral [³H]GABA    10 nM   4600 nM GABA   30 min Scintillation 214 (antagonist cortex (+10 μM (1 mM) RT counting radioligand) isoguvacine) (+10 μM baclofen) choline transporter human [³H)hemicholinium-3     3 nM    3.9 nM hemicholinium-   60 min Scintillation 648 (CHT1) (h) recombinant 3 RT counting (antagonist (CHO cells) (10 μM) radioligand) 5-HT transporter (h) human [³H]imipramine     2 nM    1.7 nM imipramine   60 min Scintillation 566 (antagonist recombinant (10 μM) RT counting radioligand) (CHO cells) Other enzymes MAO-A (antagonist rat cerebral [³H]Ro    10 nM     14 nM clorgyline   60 min Scintillation 36 radioligand) cortex 41-1049 (1 μM) 37° C. counting

Table 36 shows enzyme and uptake assay conditions.

TABLE 36 Enzyme and Uptake Assay Conditions Substrate/ Measured Detection Assay Source Stimulus/Tracer Incubation Component Method Bibl. Kinases Abl kinase (h) human recombinant ATP + Ulight-TK  60 min phospho-Ulight-TK peptide LANCE 556 (insect cells) peptide RT (100 nM) CaMK2α (h) human recombinant ATP + Ulight-  30 min phospho-Ulight- LANCE 647 CGSGSGRPRTSSF AEG RT CGSGSGRPRTSSF AEG (50 nM) CDK2 (h) human recombinant ATP + Ulight-  30 min phospho-Ulight- LANCE 469 (cycA) CFFKNIVTPRTPPP RT CFFKNIVTPRTPPP SQGK-amide (50 nM) SQGK-amide ERK2 (h) human recombinant ATP + Ulight-  15 min phospho-Ulight- LANCE 671 (P42^(mapk)) (E. coli) CFFKNIVTPRTPPP RT CFFKNIVTPRTPPP SQGK-amide (100 nM) SQGK-amide FLT-1 kinase (h) human recombinant ATP + Ulight-TK  15 min phospho-Ulight-TK peptide LANCE 650 (VEGFRI) (Sf9 cells) peptide RT (100 nM) Fyn kinase (h) human recombinant ATP + biotinyl-βAβAβ  60 min phospho-biotinyl-βAβAβ HTRF 626 (insect cells) AYQAEENTYDEYEN RT AYQAEENTYDEYEN (2 μM) IRK (h) human recombinant ATP + Ulight-Poly  10 min phospho-Ulight-Poly LANCE 467 (InsR) GAT[EAY(1:1:1)]n RT GAT[EAY(1:1:1)]n (50 nM) Lyn A kinase (h) human recombinant ATP + biotinyl-βAβAβ 120 min phospho-biotinyl-βAβAβ HTRF 41 (insect cells) AKVEKIGEGTYGVVYK RT AKVEKIGEGTYGVV (400 nM) YK p38a kinase (h) human recombinant ATP + Ulight-  60 min phospho-Ulight- LANCE 620 (E. coli) CFFKNIVTPRTPPP RT CFFKNIVTPRTPPP SQGK-amide SQGK-amide (100 nM) ZAP70 kinase (h) human recombinant ATP + biotinyl-  15 min phospho-biotinyl-βAβAβ HTRF 556 (insect cells) βAβAβ RT ADEEEYFIPP ADEEEYFIPP (2 μM) Other enzymes COX1(h) human recombinant Arachidonic acid (3 μM) +   3 min Resorufin (oxydized Fluorimetry 1480 ADHP (25 μM) RT ADHP) COX2(h) human recombinant Arachidonic acid (2 μM) +   5 min Resorufin (oxydized Fluorimetry 1480 (Sf9 cells) ADHP (25 μM) RT ADHP) 5-lipoxygenase (h) human recombinant arachidonic acid  20 min rhodamine 123 Fluorimetry 1068 (Sf9 cells) (cytosol) (25 μM) RT 12-lipoxygenase (h) human platelets arachidonic acid   5 min ferric oxidation Photometry 472 (4 μM) RT of xylenol orange inducible NOS mouse recombinant L-arginine 120 min NO₂ Photometry 236 (E. coli) (100 μM) 37″C PDE2A1 (h) human recombinant [3H]cAMP + cAMP (2 μM)  20 min [3H]5′AMP Scintillation 1399 (Sf9 cells) RT counting PDE3B (h) human recombinant [3H]cAMP + cAMP  20 min [3H]5′AMP Scintillation 1399 (Sf9 cells) (0.5 μM) RT counting PDE4D2 (h) human recombinant [3H]cAMP + cAMP  20 min [³H]5′AMP Scintillation 1399 (Sf9 cells) (0.5 μM) RT counting PDE5 (h) human platelets [³H]cGMP +  60 min [3H]5′GMP Scintillation 263 (non-selective) cGMP (1 μM) RT counting PDE6 bovine retina [³H]cGMP +  60 min [3H]5'GMP Scintillation 306 (non-selective) cGMP (2 μM) RT counting ACE (h) human recombinant Abz-FRK(Dnp)-P-OH  30 min Abz-Phe-Arg Fluorimetry 1128 (15 μM) 37° C. ACE-2 (h) human recombinant Mca-Tyr-Val-Ala-Asp-  20 min Mca peptides Fluorimetry 802 (murine cells) Pro-Ala-Lys-(DNP)-OH RT (10 μM) BACE-1 (h) human recombinant Mca-S-E-V-N-L-D-A-E-F  60 min Mca-S-E-V-N-L—NH₂ Fluorimetry 462 (β-secretase) (murine cells) R-K(Dnp)-R-R—NH₂ RT (6 μM) caspase-3 (h) human recombinant benzyloxycarbonyl-Asp-  60 min AFC Fluorimetry 476 (E. coli) Glu-Val-Asp-AFC RT (3.6 μM) caspase-8 (h) human recombinant benzyloxycarbonyl-Ile-  45 min AFC Fluorimetry 408 (E. coli) Glu-Thr-Asp-AFC (10 μM) 37° C. HIV-1 protease protein viral antranilyl-HIV (75 μM)  40 min N-terminal tripeptide Fluorimetry 244 recombinant (E. coli) 37° C. MMP-1 (h) human recombinant DNP-Pro-Cha-Gly-  40 min Cys(Me)-His-Ala- Fluorimetry 342 (E. coli) Cys(Me)-His-Ala-Lys(n- 37° C. Lys(n-Me-Abz)-NH₂ Me-Abz)-NH₂ (10 μM) MMP-2 (h) human recombinant NFF-2  90 min Mca-Arg-Pro-Lys- Fluorimetry 297 (10 μM) 37° C. Pro-Tyr-Ala MMP-9 (h) human recombinant NFF-2  90 min Mca-Arg-Pro-Lys- Fluorimetry 297 (10 μM) 37° C. Pro-Tyr-Ala guanylyl cyclase (h) human recombinant GTP 10 min cGMP HTRF 1076 (activator effect) (10 μM) RT (100 μM SNP for control) acetylcholinesterase human recombinant Acetylthiocholine (400 μM)  30 min 5 thio 2 nitrobenzoic acid Photometry 63 (h) (HEK-293 cells) RT COMT (catechol-0- porcine liver esculetin  30 min scopoletin Fluorimetry 519 methyl transferase) (1 μM) 37° C. MAO-B (h) human recombinant D-Luciferin  60 min methyl ester luciferin Luminescence 1134 recombinant derivative 37° C. enzyme (4 μM) xanthine oxidase/ purified xanthine hypoxanthine (10 μM)  10 min O₂ ⁻+ uric acid Photometry 153 superoxide 02- oxidase RT scavenging from bovine milk ATPase(Na⁺/K⁺) porcine cerebral ATP  60 min Pi Photometry 71 cortex (2 mM) 37° C. Peptidase, Human Raji cells Glutaryl-Ala-Ala-Phe-4-  30 min Glutaryl-Ala-Ala- Photometry 1352, Metalloproteinase, methoxy-2-naphthylamide 37° C. Phe-4-Methoxy-2- 1353 Neutral Endopeptidase naphthylamide -->4- Methoxy-2- naphthylamine

Analysis and Expression of Results

In Vitro Pharmacology: Binding Assays

The results are expressed as a percent of control specific binding: (Measured specific binding/Control specific binding)×100; and as a percent inhibition of control specific binding: 100−((Measured specific binding/Control specific binding)×100) obtained in the presence of NS2-D6.

The IC₅₀ values (concentration causing a half-maximal inhibition of control specific binding) and Hill coefficients (nH) were determined by non-linear regression analysis of the competition curves generated with mean replicate values using Hill equation curve fitting:

$Y = {D + \left\lbrack \frac{A - D}{1 + \left( {C/C_{50}} \right)^{nH}} \right\rbrack}$ where Y=specific binding, A=left asymptote of the curve, D=right asymptote of the curve, C=compound concentration, C₅₀=IC₅₀, and nH=slope factor. This analysis was performed using software developed at Cerep (Hill software) and validated by comparison with data generated by the commercial software SigmaPlot® 4.0 for Windows® (© 1997 by SPSS Inc.). The inhibition constants (K_(i)) were calculated using the Cheng Prusoff equation:

$K_{i} = \frac{{IC}_{50}}{\left( {1 + {L/K_{D}}} \right)}$ where L=concentration of radioligand in the assay, and KD=affinity of the radioligand for the receptor. A scatchard plot is used to determine the KD.

In Vitro Pharmacology: Enzyme and Uptake Assays

The results are expressed as a percent of control specific activity: (Measured specific activity/Control specific activity)×100; and as a percent inhibition of control specific activity: 100−((Measured specific activity/Control specific activity)×100) obtained in the presence of NS2-D6.

The IC₅₀ values (concentration causing a half-maximal inhibition of control specific activity), EC₅₀ values (concentration producing a half-maximal increase in control basal activity), and Hill coefficients (nH) were determined by non-linear regression analysis of the inhibition/concentration-response curves generated with mean replicate values using Hill equation curve fitting:

$Y = {D + \left\lbrack \frac{A - D}{1 + \left( {C/C_{50}} \right)^{nH}} \right\rbrack}$ where Y=specific activity, A=left asymptote of the curve, D=right asymptote of the curve, C=compound concentration, C₅₀=IC₅₀ or EC₅₀, and nH=slope factor. This analysis was performed using software developed at Cerep (Hill software) and validated by comparison with data generated by the commercial software SigmaPlot® 4.0 for Windows® (© 1997 by SPSS Inc.).

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Tolleson (2012),     Hindawi Publishing Corporation, Enzyme Research, Volume 2012,     Article ID 416062, 7. 

We claim:
 1. A method of treating a disease, disorder, or condition in which aldehyde toxicity is implicated in a subject in need thereof, comprising administering topically or systemically to the subject a compound of Formula II-A or II-B:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is selected from —NH₂, —NHD, or —ND₂; R² is selected from hydrogen or deuterium; R³ is —CD₃; R⁴ is —CD₃; and R⁵, R⁶, R⁷, and R⁸ are each independently selected from hydrogen or deuterium.
 2. The method of claim 1, wherein at least one of R², R⁵, R⁶, R⁷, or R⁸ is deuterium.
 3. The method of claim 1, wherein the compound is of Formula II-A, or a pharmaceutically acceptable salt thereof.
 4. The method of claim 3, wherein at least one of R², R⁵, R⁶, R⁷, or R⁸ is deuterium.
 5. The method of claim 1, wherein each of R⁵, R⁶, R⁷, and R⁸ is as defined in an entry set forth in the table below: TABLE 1c Entry R⁵ R⁶ R⁷ R⁸ i H H H H ii H H H D iii H H D H iv H D H H v D H H H vi H H D D vii H D H D viii D H H D ix H D D H x D H D H xi D D H H xii H D D D xiii D H D D xiv D D H D xv D D D H xvi D D D D

or a pharmaceutically acceptable salt thereof.
 6. The method of claim 1, wherein the compound is of the following structure:

or a pharmaceutically acceptable salt thereof.
 7. The method of claim 1, wherein each position of deuterium enrichment in the compound comprises deuterium in an amount of about 50% or greater.
 8. The method of claim 1, wherein each position of deuterium enrichment in the compound comprises deuterium in an amount of 90% or greater.
 9. The method of claim 6, wherein each position of deuterium enrichment in the compound comprises deuterium in an amount of about 50% or greater.
 10. The method of claim 6, wherein each position of deuterium enrichment in the compound comprises deuterium in an amount of about 90% or greater.
 11. The method of claim 6, wherein each position of deuterium enrichment in the compound comprises deuterium in an amount of 95% or greater.
 12. The method of claim 1, wherein the disease, disorder, or condition is an ocular disorder.
 13. The method of claim 12, wherein the ocular disorder is macular degeneration or Stargardt disease.
 14. The method of claim 12, wherein the ocular disorder is selected from the group consisting of dry eye syndrome, cataracts, keratoconus, bullous and other keratopathy, Fuch's endothelial dystrophy, allergic conjunctivitis, ocular cicatricial pemphigoid, conditions associated with PRK healing and other corneal healing, conditions associated with tear lipid degradation or lacrimal gland dysfunction, uveitis, scleritis, ocular Stevens Johnson Syndrome, and ocular rosacea.
 15. The method of claim 14, wherein the ocular disorder is dry eye syndrome.
 16. The method of claim 14, wherein the ocular disorder is selected from the group consisting of uveitis, scleritis, ocular Stevens Johnson Syndrome, and ocular rosacea.
 17. The method of claim 14, wherein the ocular disorder is ocular rosacea or uveitis.
 18. The method of claim 1, wherein the disease, disorder, or condition is a skin disease, disorder, or condition selected from the group consisting of psoriasis, topical (discoid) lupus, contact dermatitis, atopic dermatitis, allergic dermatitis, radiation dermatitis, acne vulgaris, and Sjogren-Larsson Syndrome and other ichthyoses.
 19. The method of claim 18, wherein the skin disease, disorder, or condition is contact dermatitis, atopic dermatitis, allergic dermatitis, or radiation dermatitis.
 20. The method of claim 18, wherein the skin disease, disorder, or condition is Sjogren-Larsson Syndrome.
 21. The method of claim 9, wherein the disease, disorder, or condition is an autoimmune, immune-mediated, inflammatory, cardiovascular, or neurological disease, or diabetes, metabolic syndrome, or a fibrotic disease.
 22. The method of claim 1, wherein the disease, disorder, or condition is selected from the group consisting of lupus, scleroderma, asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, inflammatory bowel disease, sepsis, atherosclerosis, ischemic-reperfusion injury, Parkinson's disease, Alzheimer's disease, succinic semialdehyde dehydrogenase deficiency, multiple sclerosis, and amyotrophic lateral sclerosis. 